Micro-RNA family that modulates fibrosis and uses thereof

ABSTRACT

The present invention relates to the identification of a microRNA family, designated miR-29a-c, that is a key regulator of fibrosis in cardiac tissue. The inventors show that members of the miR-29 family are down-regulated in the heart tissue in response to stress, and are up-regulated in heart tissue of mice that are resistant to both stress and fibrosis. Also provided are methods of modulating expression and activity of the miR-29 family of miRNAs as a treatment for fibrotic disease, including cardiac hypertrophy, skeletal muscle fibrosis other fibrosis related diseases and collagen loss-related disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/671,445, filed Jan. 29, 2010, which is a national stage applicationof International Application No. PCT/US2008/071839, filed Jul. 31, 2008,which claims the benefit of U.S. Provisional Application No. 60/952,917,filed Jul. 31, 2007; U.S. Provisional Application No. 60/980,303, filedOct. 16, 2007, and U.S. Provisional Application No. 61/047,014, filedApr. 22, 2008, all of which are herein incorporated by reference intheir entireties.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with grant support under grant no. HL53351-06from the National Institutes of Health. The government has certainrights in the invention.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:MIRG_(—)005_(—)04US_SeqList_ST25, date recorded: Mar. 15, 2013 file size5 kilobytes).

FIELD OF THE INVENTION

The present invention relates generally to the fields of developmentalbiology and molecular biology. More particularly, it concerns generegulation and cellular physiology in fibroblasts by the miR-29 family.This miRNA family plays an important role in collagen deposition,particularly collagen deposition mediated by fibroblasts.

BACKGROUND OF THE INVENTION

Heart disease and its manifestations, including coronary artery disease,myocardial infarction, congestive heart failure and cardiac hypertrophy,clearly presents a major health risk in the United States today. Thecost to diagnose, treat and support patients suffering from thesediseases is well into the billions of dollars. Two particularly severemanifestations of heart disease are myocardial infarction and cardiachypertrophy. With respect to myocardial infarction, typically an acutethrombocytic coronary occlusion occurs in a coronary artery as a resultof atherosclerosis and causes myocardial cell death. Becausecardiomyocytes, the heart muscle cells, are terminally differentiatedand generally incapable of cell division, they are generally replaced byscar tissue when they die during the course of an acute myocardialinfarction. Scar tissue is not contractile, fails to contribute tocardiac function, and often plays a detrimental role in heart functionby expanding during cardiac contraction, or by increasing the size andeffective radius of the ventricle, for example, becoming hypertrophic.Although initial collagen deposition is required for infarct healing andto prevent cardiac rupture, the continuous production of collagen byfibroblasts induces interstitial fibrosis surrounding the myocytes inthe infarct borderzone and remote myocardium of the infracted heart.This fibrosis induces stiffness, diastolic dysfunction, andcardiomyocyte hypertrophy due to the increase in stress and can alsolead to arrythmias.

Cardiac hypertrophy is an adaptive response of the heart to virtuallyall forms of cardiac disease, including those arising from hypertension,mechanical load, myocardial infarction, cardiac arrhythmias, endocrinedisorders, and genetic mutations in cardiac contractile protein genes.While the hypertrophic response is initially a compensatory mechanismthat augments cardiac output, sustained hypertrophy can lead to dilatedcardiomyopathy (DCM), heart failure, and sudden death. In the UnitedStates, approximately half a million individuals are diagnosed withheart failure each year, with a mortality rate approaching 50%. Thecauses and effects of cardiac hypertrophy have been extensivelydocumented, but the underlying molecular mechanisms have not beenelucidated. Understanding these mechanisms is a major concern in theprevention and treatment of cardiac disease and will be crucial as atherapeutic modality in designing new drugs that specifically targetcardiac hypertrophy and cardiac heart failure.

Treatment with pharmacological agents represents the primary mechanismfor reducing or eliminating the manifestations of heart failure.Diuretics constitute the first line of treatment for mild-to-moderateheart failure. If diuretics are ineffective, vasodilatory agents, suchas angiotensin converting enzyme (ACE) inhibitors (e.g., enalopril andlisinopril) or inotropic agent therapy (i.e., a drug that improvescardiac output by increasing the force of myocardial muscle contraction)may be used. Unfortunately, many of these standard therapies havenumerous adverse effects and are contraindicated in some patients. Thus,the currently used pharmacological agents have severe shortcomings inparticular patient populations. The availability of new, safe andeffective agents would undoubtedly benefit patients who either cannotuse the pharmacological modalities presently available, or who do notreceive adequate relief from those modalities.

Cardiac myocytes are normally surrounded by a fine network of collagenfibers. In response to pathological stress, cardiac fibroblasts andextracellular matrix proteins accumulate disproportionately andexcessively. Myocardial fibrosis, a characteristic of all forms ofpathological hypertrophy, leads to mechanical stiffness, whichcontributes to contractile dysfunction (Abraham et al., 2002). Anotherhallmark of pathological hypertrophy and heart failure is there-activation of a set of fetal cardiac genes, including those encodingatrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) andfetal isoforms of contractile proteins, such as skeletal α-actin andβ-myosin heavy chain (MHC). These genes are typically repressedpost-natally and replaced by the expression of a set of adult cardiacgenes (McKinsey and Olson, 2005). The consequences of fetal geneexpression on cardiac function and remodeling (e.g., fibrosis) are notcompletely understood. However, the up-regulation of β-MHC, a slowATPase, and down-regulation of α-MHC, a fast contracting ATPase, inresponse to stress has been implicated in the diminution of cardiacfunction (Bartel, 2004) and BNP is known to play a dominant role incardiac fibrosis.

In addition to cardiac fibrosis, there are a number of disorders orconditions that are associated with fibrosis of various tissues.Congenital hepatic fibrosis, an autosomal recessive disease, is a raregenetic disease that affects both the liver and kidneys. The disease ischaracterized by liver abnormalities, such as hepatomegaly, portalhypertension, and fiber-like connective tissue that spreads over andthrough the liver (hepatic fibrosis). Pulmonary fibrosis, or scarring ofthe lung, results from the gradual replacement of normal lung air sacswith fibrotic tissue. When the scar forms, the tissue becomes thicker,causing an irreversible loss of the tissue's ability to transfer oxygeninto the bloodstream. The most current thinking is that the fibroticprocess in pulmonary tissue is a reaction (predisposed by genetics) tomicroscopic injury to the lung. While the exact cause remains unknown,associations have been made with inhaled environmental and occupationalpollutants, cigarette smoking, diseases such as scleroderma, rheumatoidarthritis, lupus and sarcoidosis, certain medications and therapeuticradiation.

Scleroderma is a chronic disease characterized by excessive deposits ofcollagen in the skin or other organs. The localized type of the disease,while disabling, tends not to be fatal. The systemic type or systemicsclerosis, which is the generalized type of the disease, can be fatal asa result of heart, kidney, lung or intestinal damage. Sclerodermaaffects the skin, and in more serious cases it can affect the bloodvessels and internal organs.

Skeletal muscle fibrosis is a phenomenon which frequently occurs indiseased or damaged muscle. It is characterized by the excessive growthof fibrous tissue which usually results from the body's attempt torecover from injury. Fibrosis impairs muscle function and causesweakness. The extent of loss of muscle function generally increases withthe extent of fibrosis. Victims of muscular dystrophies, particularlyBecker muscular dystrophy (BMD) and the more severely penetratingallelic manifestation, Duchenne muscular dystrophy (DMD), frequentlysuffer from increasing skeletal muscle fibrosis as the diseaseprogresses. Other afflictions such as denervation atrophy are known toproduce skeletal muscle fibrosis, as well as neuromuscular diseases,such as acute polyneuritis, poliomyelitis, Werdig/Hoffman disease,amyotrophic lateral sclerosis (Lou Gehrig's Disease), and progressivebulbar atrophy disease.

MicroRNAs have recently been implicated in a number of biologicalprocesses including regulation of developmental timing, apoptosis, fatmetabolism, and hematopoietic cell differentiation among others.MicroRNAs (miRs) are small, non-protein coding RNAs of about 18 to about25 nucleotides in length that are derived from individual miRNA genes,from introns of protein coding genes, or from poly-cistronic transcriptsthat often encode multiple, closely related miRNAs. See review ofCarrington et al. (2003). MiRs act as repressors of target mRNAs bypromoting their degradation, when their sequences are perfectlycomplementary, or by inhibiting translation, when their sequencescontain mismatches.

miRNAs are transcribed by RNA polymerase II (pol II) or RNA polymeraseIII (pol III; see Qi et al. (2006) Cellular & Molecular Immunology Vol.3:411-419) and arise from initial transcripts, termed primary miRNAtranscripts (pri-miRNAs), that are generally several thousand baseslong. Pri-miRNAs are processed in the nucleus by the RNase Drosha intoabout 70- to about 100-nucleotide hairpin-shaped precursors(pre-miRNAs). Following transport to the cytoplasm, the hairpinpre-miRNA is further processed by Dicer to produce a double-strandedmiRNA. The mature miRNA strand is then incorporated into the RNA-inducedsilencing complex (RISC), where it associates with its target mRNAs bybase-pair complementarity. In the relatively rare cases in which a miRNAbase pairs perfectly with an mRNA target, it promotes mRNA degradation.More commonly, miRNAs form imperfect heteroduplexes with target mRNAs,affecting either mRNA stability or inhibiting mRNA translation.

The 5′ portion of a miRNA spanning bases 2-8, termed the ‘seed’ region,is especially important for target recognition (Krenz and Robbins, 2004;Kiriazis and Krania, 2000). The sequence of the seed, together withphylogenetic conservation of the target sequence, forms the basis formany current target prediction models. Although increasinglysophisticated computational approaches to predict miRNAs and theirtargets are becoming available, target prediction remains a majorchallenge and requires experimental validation. Ascribing the functionsof miRNAs to the regulation of specific mRNA targets is furthercomplicated by the ability of individual miRNAs to base pair withhundreds of potential high and low affinity mRNA targets and by thetargeting of multiple miRNAs to individual mRNAs. Enhanced understandingof the functions of miRNAs will undoubtedly reveal regulatory networksthat contribute to normal development, differentiation, inter- andintra-cellular communication, cell cycle, angiogenesis, apoptosis, andmany other cellular processes. Recently, the inventors reported acardiac-specific microRNA, miR-208, which is encoded by an intron of theα-myosin heavy chain (MHC) gene, and is required for up-regulation ofβ-MHC expression in response to cardiac stress and for repression offast skeletal muscle genes in the heart (see co-pending applicationWO2008/016924, which is herein incorporated by reference in itsentirety). The present invention expands on the involvement of microRNAsin the heart as well as other tissues.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that the miR-29 family,which is down-regulated in the heart in response to stress, regulatescollagen deposition and the development of fibroses, including cardiacfibrosis. Up-regulation of miR-29a-c expression or function results inthe decrease of expression of collagen and fibrin genes leading toreduced cardiac fibrosis. Accordingly, the present invention provides amethod of treating cardiac fibrosis, cardiac hypertrophy, or heartfailure in a subject in need thereof comprising identifying a subjecthaving cardiac fibrosis, cardiac hypertrophy or heart failure; andadministering to said subject an agonist of miR-29a-c expression orfunction. In one embodiment, the agonist of miR-29a-c is apolynucleotide comprising the mature sequence of miR-29a, miR-29b,miR-29c, or combinations thereof. The agonist of miR-29a-c may beadministered by parenteral administration (e.g. intravenous orsubcutaneous), oral, transdermal, sustained release, controlled release,delayed release, suppository, catheter or sublingual administration. Inanother embodiment, the method further comprises administering to thesubject a second therapy. The second therapy is selected from the groupconsisting of a beta blocker, an ionotrope, a diuretic, ACE-I, AIIantagonist, BNP, a Ca⁺⁺-blocker, an endothelin receptor antagonist, andan HDAC inhibitor.

The present invention also provides a method of preventing pathologichypertrophy or heart failure in a subject in need thereof comprisingidentifying a subject at risk of developing pathologic cardiachypertrophy or heart failure; and promoting the expression or activityof miR-29a-c in cardiac cells of said subject. In one embodiment, thepromoting expression or activity of miR-29a-c comprises delivering tothe cardiac cells an agonist of miR-29a-c or an expression vectorencoding miR-29a-c. In another embodiment, the subject at risk exhibitsone or more risk factors selected from the group consisting of longstanding uncontrolled hypertension, uncorrected valvular disease,chronic angina, recent myocardial infarction, congenital predispositionto heart disease, and pathological hypertrophy. In another embodiment,the subject at risk has been diagnosed as having a geneticpredisposition to cardiac hypertrophy. In still another embodiment, thesubject at risk has a familial history of cardiac hypertrophy.

The present invention also encompasses a transgenic, non-human mammal,the cells of which fail to express a functional miR-29a, miR29b, and/ormiR29c. In another embodiment, the invention provides a transgenic,non-human mammal, the cells of which comprise a miR-29a-c coding regionunder the control of a heterologous promoter active in the cells of saidnon-human mammal. The transgenic mammal may be a mouse.

In one embodiment, the present invention provides a method of treatingmyocardial infarction in a subject in need thereof comprising promotingexpression or activity of miR-29a-c in cardiac cells of said subject. Inanother embodiment, the present invention provides a method ofpreventing cardiac hypertrophy and dilated cardiomyopathy in a subjectin need thereof comprising promoting expression or activity of miR-29a-cin cardiac cells of said subject. In another embodiment, the presentinvention provides a method of inhibiting progression of cardiachypertrophy in a subject in need thereof comprising promoting expressionor activity of miR-29a-c in cardiac cells of said subject.

The present invention also contemplates a method of treating orpreventing a tissue fibrosis in a subject comprising identifying asubject having or at risk of tissue fibrosis; and increasing theexpression and/or activity of miR-29a-c in skeletal muscle or fibroblastcells of the subject. The tissue fibrosis may be cardiac fibrosis,scleroderma, skeletal muscle fibrosis, hepatic fibrosis, kidneyfibrosis, pulmonary fibrosis, or diabetic fibrosis. In some embodiments,increasing the expression and/or activity of miR-29a-c comprisesadministering an agonist of miR-29a-c to the subject. An agonist ofmiR-29a-c may be a polynucleotide comprising the sequence of a maturemiR-29a, miR-29b, and/or miR-29c sequence. The agonist of miR-29a-c mayalso be an expression vector encoding miR-29a, miR-29b, and/or miR-29c.In one embodiment, the method further comprises administering anon-miR-29a-c anti-fibrotic therapy to the subject.

The present invention also provides a method for identifying a modulatorof miR-29a-c comprising contacting a cell with a candidate compound;assessing miR-29a-c activity or expression; and comparing the activityor expression in step (b) with the activity or expression of miR-29a-cin the absence of the candidate compound, wherein a difference betweenthe measured activities or expression of miR-29a-c indicates that thecandidate compound is a modulator of miR-29. The cell may be contactedwith the candidate compound in vitro or in vivo. Suitable candidatecompounds include proteins, peptides, polypeptides, polynucleotides,oligonucleotides or small molecules.

The present invention also encompasses a pharmaceutical compositioncomprising an agonist or antagonist of miR-29a-c. In some embodiments,the pharmaceutical composition may be formulated for injection ortopical administration. The formulation for topical administration maybe a gel, cream, lotion, or ointment.

The present invention provides a method of inducing collagen depositionin a tissue comprising contacting said tissue with an antagonist ofmiR-29a-c. The antagonist may be an antagonist of miR-29a, miR-29b, ormiR-29c. The antagonist may be an antagomir of miR-29a-c, an antisenseoligonucleotide that targets a mature miR-29a-c sequence, or aninhibitory RNA molecule, such as a siRNA or shRNA, that comprises asequence identical to a mature miR-29a-c sequence, or a ribozyme oranother inhibitory nucleic acid. In one embodiment, the method furthercomprises contacting said tissue with a second agent. The second agentmay be topical vitamin A, topical vitamin C, or vitamin E. In anotherembodiment, the method further comprises subjecting said tissue to asecond treatment, such as a chemical peel, laser treatment,dermaplaning, or dermabrasion. In another embodiment, the tissue is in asubject that suffers from Ehler's-Danlos syndrome or Vitamin Cdeficiency.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be better understood by reference to one or more ofthese drawings in combination with the detailed description of specificembodiments presented herein.

FIGS. 1A-B. miR-208 is encoded by the α-MHC gene and is expressedspecifically in the heart. (FIG. 1A) miR-208 is encoded within an intronof the α-MHC gene. Asterisks indicate sequence conservation (SEQ IDNOS:1-5). (FIG. 1B) Detection of miR-208 transcripts by Northernanalysis of adult mouse tissues. U6 mRNA serves as a loading control.

FIGS. 2A-B. Regulation of α- and β-MHC. (FIG. 2A) Regulation of classswitch by thyroid hormone and TRE. (FIG. 2B) Model forstress/hypothyroidism in fast-to-slow muscle fiber contactility switch.

FIG. 3. Detection of miR-208 in human heart. Transcripts for α-MHC andmiR-208 were detected by Northern blot of cardiac tissue from six normalindividuals and six individuals with idiopathic cardiomyopathy. A closecorrelation exists between the level of expression of α-MHC andpre-miR-208, whereas mature miR-208 expression is maintained after thelatter has been down-regulated.

FIGS. 4A-B. Generation of miR-208 mutant mice. (FIG. 4A) Strategy togenerate miR-208 mutant mice by homologous recombination. The pre-miRNAsequence (located within intron 29 of the mouse α-MHC gene in mosttranscripts) was replaced with a neomycin resistance cassette flanked byloxP sites. The neomycin cassette was removed in the mouse germline bybreeding heterozygous mice to transgenic mice harboring the CAG-Cretransgene. (FIG. 4B) Detection of miR-208 transcripts by Northernanalysis of hearts from wild-type and miR-208 mutant mice.

FIG. 5. Western analysis of α-MHC and β-MHC protein levels in hearts ofneonatal mice of the indicated genotypes. Two mice of each genotype wereanalyzed. GAPDH was detected as a loading control.

FIG. 6. MiR-208^(−/−) mice show reduced cardiac hypertrophy in responseto pressure overload. Histological sections of hearts of wild-type andmiR-208^(−/−) mice stained for Masson trichrome. The absence of miR-208diminishes hypertrophy and fibrosis seen in wild-type mice subjected tothoracic aortic banding (TAB) for 21 days. Scale bar equals 2 mm in toppanel and 20 μm for bottom panel.

FIG. 7. MiR-208^(−/−) mice show reduced cardiac hypertrophy in responseto calcineurin activation. Histological sections of hearts of 6 week-oldmice expressing a calcineurin transgene (CnA-Tg) and hearts ofmiR-208^(−/−); CnA-Tg stained for Masson trichrome. Absence of miR-208diminishes hypertrophy and fibrosis seen in CnA-Tg mice. Scale bar=2 mmfor top panel, 20 μm for bottom panel.

FIG. 8. Mir-208^(−/−) mice fail to up-regulate β-MHC in response to TABand calcineurin activation.

FIG. 9. Western analysis of α and β-MHC protein levels in adultwild-type and miR-208 transgenic animals. GAPDH was detected as aloading control.

FIG. 10. miR-208^(−/−) mice fail to up-regulate β-MHC in response tohypothyroidism with PTU treatment.

FIG. 11. Schematic diagram of the role of miR-208 in the control ofβ-MHC expression.

FIG. 12. Schematic diagram of the role of miR-208 in the regulation ofβ-MHC and fast skeletal muscle gene expression via Thrap1.

FIG. 13. Mechanisms of action of microRNAs during cardiac hypertrophy.

FIGS. 14A-C. miRNA expression during cardiac hypertrophy and remodeling.(FIG. 14A) H&E stained sections of representative hearts from micefollowing sham and TAB for 21 days and from CnA Tg mice. Scale barequals 2 mm. (FIG. 14B) Venn diagrams showing numbers of microRNAs thatchanged in expression in each type of heart are shown below. (FIG. 14C)Northern blots of microRNAs that change in expression duringhypertrophy. U6 RNA was detected as a loading control.

FIG. 15. miR-29a-c expression is down-regulated in response to cardiacstress. Hearts from wild-type mice (WT) and mice with hypertrophy andfibrosis induced by a calcineurin transgene (CnA) or TAB are shown onthe left. The relative level of expression of miR-29a-c in each type ofheart is shown on the right.

FIG. 16. Microarray analysis of hearts from miR-208 knockout micecompared to wild-type. Microarray analysis was performed on mRNAisolated from wild-type and miR-208-null hearts at 6 weeks of age. Themost down-regulated miRNA, next to miR-208, is miR-499.

FIG. 17. miR-29 family is dramatically up-regulated in miR-208-nullhearts.

FIG. 18. miR-29 family targets mRNAs encoding collagens and othercomponents of the extracellular matrix involved in fibrosis. Based ontheir high sequence homology, the miR 29 family consists of 4 members;miR-29a, miR29b-1 and -2 and miR-29c. The sequences of the mature miRNAsare shown (SEQ ID NOS:18-20). The mature sequences of miR-29b-1 andmiR-29b-2 are identical. Together this family is directed against manycomponents of the extracellular matrix involved in fibrosis.

FIG. 19. Model for the control of cardiac fibrosis by miR-208 and miR-29family. In the normal heart, miR-208 inhibits the expression ofmiR-29a-c. In the absence of miR-208, miR-29a-c expression isup-regulated, preventing the expression of extracellular matrix andfibrosis in response to stress. The functions of miR-208, miR-499 andmiR-29 are interlinked. Loss of miR-208 can be cardioprotective bypreventing expression of miR-499 and up-regulating expression ofmiR-29a-c, with consequent blockade to fibrosis.

FIGS. 20A-D. miR-29a-c regulates the expression of extracellular matrixproteins. (FIG. 20A) Potential binding sites for miR-29a-c in 3′ UTRregions of key fibrotic genes. (FIG. 20B) Real-time PCR analysis ofpredicted target genes in both the borderzone and remote myocardium 3days after MI, shows a decrease in miR-29a-c to correlate to an increasein collagens (COL1A1, COL1A2 and COL3A1) and fibrillin (FBN1), whilethere was no significant change in elastin (ELN1). (FIG. 20C) Northernblot analysis on COS cells transfected with increasing amounts of theCMV expression plasmid encoding the miR-29b-1/miR-29a cluster, showsefficient overexpression of miR-29a-b. The top band corresponds to thepre-miRNA, while the lower band corresponds to the mature miRNA. (FIG.20D) Luciferase experiments using the endogenous UTR sequences of thepredicted target genes, showing miR-29a-c to repress expression ofluciferase in response to increasing amounts of miR-29a-c while thisdecrease was absent when using an unrelated miR, miR-206.

FIGS. 21A-B. miR-29a-c expression responsive to TGFβ. (FIG. 21A)Real-time PCR analysis indicates that all three miR-29 family membersare downregulated in fibroblasts in response to TGFβ. (FIG. 21B)Northern analysis showing miR-29a-c expression is upregulated in miR-208mutant animals which coincides with an increase in BNP expression asdetermined by real-time PCR.

FIGS. 22A-G. miR-29a-c inhibition induces fibrosis in vivo. (FIG. 22A)Chemical structure of anti-miR-29a-c and mismatch (mm) miR-29a-c. (FIG.22B) Northern blot analysis showing tissue specific knockdown after 3days in response to intravenous injection of 80 mg/kg of eitheranti-miR-29a-c or mm miR-29a-c or a comparable volume of saline. (FIG.22C) Real-time PCR analysis of liver extracts indicate a pronouncedincrease in collagen expression in response to miR-29a-c knockdown,while this effect was absent after saline or mm injection. (FIG. 22D)Tissue collection, three weeks after intravenous injection with 80 mg/kgon two consecutive days of either anti-miR-29a-c or mm miR-29a-coligonucleotide or a comparable volume of saline, indicates a severeknockdown of miR-29a-c in heart, liver and kidney, while miR-29a-clevels in lungs appear unaffected. (FIG. 22E) Real-time PCR analysis ofheart extracts indicate a increase in cardiac collagen expression inresponse to miR-29a-c knockdown. (FIG. 22F) Real-time PCR analysisindicating an increase in miR-29b expression in fibroblasts two daysafter miR-29b mimic treatment, while miR-29a levels were unchanged andmiR-29c levels only slightly increased. (FIG. 22G) miR-29boverexpression in fibroblasts represses the expression of collagen genesas determined by real-time PCR analysis.

FIG. 23. Expression of miR-29 family members in various tissues inresponse to miR-29b knockdown. Knockdown of all miR-29 members in thedifferent tissues indicates that miR-29b shows a 50% reduction in theheart in response to anti-miR-29b, while miR-29a and -c only showmarginal changes. However, the knockdown of miR-29b in liver and kidneyin response to anti-miR-29b is almost complete, while miR-29a and -calso appear to be reduced in these tissues in response to anti-miR-29b.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Cardiac and skeletal muscles respond to a variety of pathophysiologicalstimuli such as workload, thyroid hormone signaling and injury bymodulating the expression of myosin isoforms, which regulate theefficiency of contraction. Recently, the inventors reported acardiac-specific microRNA, miR-208, which is encoded by an intron of theα-myosin heavy chain (MHC) gene, and is required for up-regulation ofβ-MHC expression in response to cardiac stress and for repression offast skeletal muscle genes in the heart (see co-pending applicationWO2008/016924, which is herein incorporated by reference in itsentirety).

Here, the inventors extend their earlier work and show that miR-208 alsodown-regulates a family of related miRNAs, miR-29a-c. Because miR-29a-care expressed ubiquitously, and are involved in regulation of collagendeposition, strategies to upregulate miR-29a-c expression haveapplications in the prevention of a variety of tissue fibroses includingcardiac fibrosis, as well as skeletal muscle, liver, pulmonary, diabeticand kidney fibrosis. Regulation of miR-29a-c in cardiac cells, such ascardiac fibroblasts, can be used to treat or prevent cardiac hypertrophyor heart failure in a subject. Thus, one aspect of the invention isagonism of miR-29a-c expression or activity, optionally in conjunctionwith inhibiting miR-208. Agonism may involve introducing exogenousmiR-29a-c into the heart or other tissues of interest, either directlyusing naked nucleic acid or a delivery vehicle such as alipid/liposome/nanoparticle, or through gene expression, for example byusing adenoviral vectors or other means of ectopic expression to reducefibrosis. Activation of the anti-fibrotic function of miR-29a-c throughpharmaceutical “small molecules” also is contemplated, as are screens toidentify such compounds.

The increase in collagen that ensues following repression of miR-29a-cexpression, for example, following myocardial infarction (MI) and otherforms of stress, may indicate another role for miR-29a-c. One focus ofthe inventors' work is cardiac fibrosis, and an examination of a subsetof key regulatory genes, namely collagen I, III, elastin and fibrillin,showed a striking increase in both collagens and fibrillin in responseto miR-29a-c downregulation, while there was no increase in elastin. Assuch, therapeutically repressing miR-29a-c to increase collagendeposition presents a unique option for addressing conditionscharacterized by loss of collagen, such as in cosmetic applications andscarring.

MicroRNA 29 (miR-29) is a family of microRNAs that consists of 4 knownmembers, miR-29a, b1 and 2 (identical) and c. While miR29b-1 and 29astem from the same transcript originating from chromosome 7 in humansand chromosome 6 in mice, the miRNA cluster containing miR29b-2 andmiR29c is transcribed from chromosome 1 in both species. The maturemiRNA sequences for each of the human miR-29 family members is listedbelow:

(SEQ ID NO: 18) hsa-miR-29a uagcaccaucugaaaucgguua (SEQ ID NO: 19)hsa-miR-29b-1 and b-2 uagcaccauuugaaaucaguguu (SEQ ID NO: 20)hsa-miR-29c uagcaccauuugaaaucgguua

These microRNAs form a family based on their sequence homology (Yu etal; 2006). Since there are only minor differences between the familymembers, and the members have a 100% conserved seed region (which helpsto define target determination), they are very likely to target the samemRNA targets (FIG. 18), and lower gene expression of these specifictarget genes. Target determination for the miR-29 family revealed thatthe miR-29 family shows a high preference for targeting genes involvedin collagen formation as well as other extracellular matrix proteins,such as elastin (ELN), fibrillin 1 (FBN1), collagen type I, α1 and α2(COL1A1, COL1A2) collagen type III, α1 (COL3A1), metallopeptidases, andintegrins. In response to pathological stress, cardiac fibroblasts andextracellular matrix proteins accumulate disproportionately andexcessively. Myocardial fibrosis, a characteristic of all forms ofpathological hypertrophy, leads to mechanical stiffness, whichcontributes to contractile dysfunction (Berk et al., 2007). Since themiR-29 family is downregulated during this remodeling process, thisfamily is likely to play an active role in the modulation of collagendeposition, and thereby regulate cardiac fibrosis and cardiaccontractility, which secondarily can induce hypertrophy and pathologicalremodeling.

As discussed previously, miR-208 appears to regulate miR-29 expressionas miR-29 is significantly up-regulated in the hearts of mice lackingboth copies of miR-208 (see Example 1). Thus, modulation of miR-208 canaffect the expression of miR-29 as well as the expression of miR-29target genes. MiR-208 is an intronic miRNA that is located within anintron of the α-MHC gene. The precise intron location is dependent onthe particular species and specific transcript. For example, in humans,miR-208 is encoded within the 28^(th) intron of the α-MHC gene, while inmice, it is encoded within the 29^(th) intron. The pre-miRNA encodingsequences for miR-208 for human, mouse, rat, and canine are provided inSEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, respectively.The mature miR-208 sequence is provided in SEQ ID NO:5. Like α-MHC,miR-208 is expressed solely in the heart. (FIG. 1).

Human pre-miR-208 (SEQ ID NO: 14)acgggcgagc ttttggcccg ggttatacct gatgctcacg tataagacga gcaaaaagct tgttggtcag aMouse pre-miR-208 (SEQ ID NO: 15)acgggtgagc ttttggcccg ggttatacct gactctcacg tataagacga gcaaaaagct tgttggtcag aRat pre-miR-208 (SEQ ID NO: 16)acgggtgagc ttttggcccg ggttatacct gactctcacg tataagacga gcaaaaagct tgttggtcag aCanine pre-miR-208 (SEQ ID NO: 17)acgcatgagc ttttggctcg ggttatacct gatgctcacg tataagacga gcaaaaagct tgttggtcag a

Using the PicTar algorithm for the identification of miRNA targets (Kreket al., 2005), the inventors identified thyroid hormone receptorassociated protein 1 (THRAP1) as a predicted target for miR-208. THRAP13′ UTR sequences from human, chimp, mouse, rat, canine, chicken, fugu,and zebrafish are provided in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13,respectively.

Human THRAP1 3′UTR (SEQ ID NO: 6)uucuugcuuu aaagcaauug gucuaaaaua uauguaaucg ucuuaauuaa aaaguugcag uaggguugcChimp THRAP1 3′UTR (SEQ ID NO: 7)uucuugcuuu aaagcaauug gucuaaaaua uauguaaucg ucuuaauuaa aacguugcag uaggguugcMouse THRAP1 3′UTR (SEQ ID NO: 8)uucuugcuuu aaagcaauug gucuaaaaua uauguaaucg ucuuaauuaa aacguugcag uaggguugcRat THRAP1 3′UTR (SEQ ID NO: 9)uucuugcuuu aaagcaauug gucuaaaaua uauguaaucg ucuuaauuaa aacguugcag uaggguugcCanine THRAP1 3′UTR (SEQ ID NO: 10)uucuugcuuu aaagcaauug gucuaaaaua uauguaaucg ucuuaauuaa aacguugcag uaggguugcChicken THRAP1 3′UTR (SEQ ID NO: 11)uucuugcuuu aaagcaauug gucuaaaaua uauguaaucg ucuuaauuaa aacguugcag uaggguugcFugu THRAP1 3′UTR (SEQ ID NO: 12)uuccugcuuu aagcaauugg uugaaaauau auguauguaa uggucuuaau uaaaaaaaca aacuaagaca aaZebrafish THRAP1 3′UTR (SEQ ID NO: 13)uuccugcuuu aaagcaauug gucuaaaaua uauguaaucg ucuucauuac aaaaacgaac caucaaacg

The present invention provides a method of treating cardiac fibrosis,cardiac hypertrophy or heart failure in a subject in need thereofcomprising identifying a subject having cardiac fibrosis, cardiachypertrophy or heart failure; and administering to the subject anagonist of miR-29 expression or function. The miR-29 agonist may be anagonist of miR-29a, miR-29b and/or miR-29c.

In one embodiment, agonists of miR-29a-c may be polynucleotidescomprising the mature miR-29a-c sequence. In some embodiments, thepolynucleotide comprises the sequence of SEQ ID NO: 18, SEQ ID NO: 19,or SEQ ID NO: 20. In another embodiment, the agonist of miR-29a-c may bea polynucleotide comprising the pri-miRNA or pre-miRNA sequence formiR-29a, miR-29b, and/or miR-29c. The polynucleotide comprising themature miR-29a-c, pre-miR-29a-c, or pri-miR-29a-c sequence may be singlestranded or double stranded. The polynucleotides may contain one or morechemical modifications, such as locked nucleic acids, peptide nucleicacids, sugar modifications, such as 2′-O-alkyl (e.g. 2′-O-methyl,2′-O-methoxyethyl), 2′-fluoro, and 4′thio modifications, and backbonemodifications, such as one or more phosphorothioate, morpholino, orphosphonocarboxylate linkages. In one embodiment, the polynucleotidecomprising a miR-29a-c sequence is conjugated to cholesterol. In anotherembodiment, the agonist of miR-29a-c may be an agent distinct frommiR-29a-c that acts to increase, supplement, or replace the function ofmiR-29a-c.

In another embodiment, the agonist of miR-29a-c may be expressed in vivofrom a vector. A “vector” is a composition of matter which can be usedto deliver a nucleic acid of interest to the interior of a cell.Numerous vectors are known in the art including, but not limited to,linear polynucleotides, polynucleotides associated with ionic oramphiphilic compounds, plasmids, and viruses. Thus, the term “vector”includes an autonomously replicating plasmid or a virus. Examples ofviral vectors include, but are not limited to, adenoviral vectors,adeno-associated virus vectors, retroviral vectors, and the like. Anexpression construct can be replicated in a living cell, or it can bemade synthetically. For purposes of this application, the terms“expression construct,” “expression vector,” and “vector,” are usedinterchangeably to demonstrate the application of the invention in ageneral, illustrative sense, and are not intended to limit theinvention.

In one embodiment, an expression vector for expressing miR-29a-ccomprises a promoter “operably linked” to a polynucleotide encodingmiR-29a, miR-29b, miR-29c, or combinations thereof. In anotherembodiment, the polynucleotide may encode the miR-29b-1/miR-29a cluster.In another embodiment, the polynucleotide may encode themiR-29b-2/miR-29c cluster. The phrase “operably linked” or “undertranscriptional control” as used herein means that the promoter is inthe correct location and orientation in relation to a polynucleotide tocontrol the initiation of transcription by RNA polymerase and expressionof the polynucleotide. The polynucleotide encoding miR-29a-c may encodethe primary-microRNA-29a-c sequence (pri-miR-29a-c), theprecursor-microRNA-29a-c sequence (pre-miR-229a-c) or the maturemiR-29a-c sequence. In another embodiment, the expression vectorcomprises a polynucleotide operably linked to a promoter, wherein saidpolynucleotide comprises the sequence of SEQ ID NO: 18. In anotherembodiment, the expression vector comprises a polynucleotide operablylinked to a promoter, wherein said polynucleotide comprises the sequenceof SEQ ID NO: 19. In still another embodiment, the expression vectorcomprises a polynucleotide operably linked to a promoter, wherein saidpolynucleotide comprises the sequence of SEQ ID NO: 20. Thepolynucleotide comprising the sequence of SEQ ID NO: 18, SEQ ID NO: 19,or SEQ ID NO: 20 may be about 18 to about 2000 nucleotides in length,about 70 to about 200 nucleotides in length, about 20 to about 50nucleotides in length, or about 18 to about 25 nucleotides in length.

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. Generally, the nucleic acidencoding a gene product is under transcriptional control of a promoter.A “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell, or introduced synthetic machinery, required toinitiate the specific transcription of a gene.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for a RNA polymerase. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription.

In other embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, rat insulin promoter, RNA pol III promoter, andglyceraldehyde-3-phosphate dehydrogenase promoter can be used to obtainhigh-level expression of the polynucleotide of interest. The use ofother viral or mammalian cellular or bacterial phage promoters which arewell-known in the art to achieve expression of a polynucleotide ofinterest is contemplated as well, provided that the levels of expressionare sufficient for a given purpose.

By employing a promoter with well-known properties, the level andpattern of expression of the polynucleotide of interest followingtransfection or transformation can be optimized. Further, selection of apromoter that is regulated in response to specific physiologic signalscan permit inducible expression of the gene product. Tables 1 and 2 listseveral regulatory elements that may be employed, in the context of thepresent invention, to regulate the expression of the gene of interest.This list is not intended to be exhaustive of all the possible elementsinvolved in the promotion of gene expression but, merely, to beexemplary thereof.

Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Promoters and enhancers are often overlapping and contiguous, oftenseeming to have a very similar modular organization.

Below is a list of viral promoters, cellular promoters/enhancers andinducible promoters/enhancers that could be used in combination with thenucleic acid encoding a gene of interest in an expression construct(Table 1 and Table 2). Additionally, any promoter/enhancer combination(as per the Eukaryotic Promoter Data Base EPDB) could also be used todrive expression of the gene. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct. In a preferredembodiment, the polynucleotide encoding the miR-29a-c or miR-29a-cantagonist is operably-linked to a fibroblast specific promoter.

TABLE 1 Promoter and/or Enhancer Promoter/Enhancer ReferencesImmunoglobulin Heavy Chain Banerji et al., 1983; Gilles et al., 1983;Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al.,1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.;1990 Immunoglobulin Light Chain Queen et al., 1983; Picard et al., 1984T-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondo et al.;1990 HLA DQ a and/or DQ β Sullivan et al., 1987 β-Interferon Goodbournet al., 1986; Fujita et al., 1987; Goodbourn et al., 1988 Interleukin-2Greene et al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin etal., 1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-DRa Shermanet al., 1989 β-Actin Kawamoto et al., 1988; Ng et al.; 1989 MuscleCreatine Kinase (MCK) Jaynes et al., 1988; Horlick et al., 1989; Johnsonet al., 1989 Prealbumin (Transthyretin) Costa et al., 1988 Elastase IOrnitz et al., 1987 Metallothionein (MTII) Karin et al., 1987; Culottaet al., 1989 Collagenase Pinkert et al., 1987; Angel et al., 1987aAlbumin Pinkert et al., 1987; Tronche et al., 1989, 1990 α-FetoproteinGodbout et al., 1988; Campere et al., 1989 t-Globin Bodine et al., 1987;Perez-Stable et al., 1990 β-Globin Trudel et al., 1987 c-fos Cohen etal., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985 Insulin Edlundet al., 1985 Neural Cell Adhesion Molecule Hirsh et al., 1990 (NCAM)α₁-Antitrypain Latimer et al., 1990 H2B (TH2B) Histone Hwang et al.,1990 Mouse and/or Type I Collagen Ripe et al., 1989 Glucose-RegulatedProteins Chang et al., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsenet al., 1986 Human Serum Amyloid A (SAA) Edbrooke et al., 1989 TroponinI (TN I) Yutzey et al., 1989 Platelet-Derived Growth Factor Pech et al.,1989 (PDGF) Duchenne Muscular Dystrophy Klamut et al., 1990 SV40 Banerjiet al., 1981; Moreau et al., 1981; Sleigh et al., 1985; Firak et al.,1986; Herr et al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wanget al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al.,1988 Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980; Katinkaet al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; deVilliers et al., 1984; Hen et al., 1986; Satake et al., 1988; Campbelland/or Villarreal, 1988 Retroviruses Kriegler et al., 1982, 1983;Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze etal., 1986; Miksicek et al., 1986; Celander et al., 1987; Thiesen et al.,1988; Celander et al., 1988; Choi et al., 1988; Reisman et al., 1989Papilloma Virus Campo et al., 1983; Lusky et al., 1983; Spandidos and/orWilkie, 1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al.,1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987Hepatitis B Virus Bulla et al., 1986; Jameel et al., 1986; Shaul et al.,1987; Spandau et al., 1988; Vannice et al., 1988 Human ImmunodeficiencyVirus Muesing et al., 1987; Hauber et al., 1988; Jakobovits et al.,1988; Feng et al., 1988; Takebe et al., 1988; Rosen et al., 1988;Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989; Braddocket al., 1989 Cytomegalovirus (CMV) Weber et al., 1984; Boshart et al.,1985; Foecking et al., 1986 Gibbon Ape Leukemia Virus Holbrook et al.,1987; Quinn et al., 1989

TABLE 2 Inducible Elements Element Inducer References MT II PhorbolEster (TFA) Palmiter et al., 1982; Heavy metals Haslinger et al., 1985;Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin etal., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTVGlucocorticoids Huang et al., 1981; Lee et (mouse mammary al., 1981;Majors et al., tumor virus) 1983; Chandler et al., 1983; Ponta et al.,1985; Sakai et al., 1988 β-Interferon poly(rI)x Tavernier et al., 1983poly(rc) Adenovirus 5 E2 ElA Imperiale et al., 1984 Collagenase PhorbolEster (TPA) Angel et al., 1987a Stromelysin Phorbol Ester (TPA) Angel etal., 1987b SV40 Phorbol Ester (TPA) Angel et al., 1987b Murine MX GeneInterferon, Newcastle Hug et al., 1988 Disease Virus GRP78 Gene A23187Resendez et al., 1988 α-2-Macroglobulin IL-6 Kunz et al., 1989 VimentinSerum Rittling et al., 1989 MHC Class I Interferon Blanar et al., 1989Gene H-2κb HSP70 ElA, SV40 Large T Taylor et al., 1989, 1990a, Antigen1990b Proliferin Phorbol Ester-TPA Mordacq et al., 1989 Tumor NecrosisPMA Hensel et al., 1989 Factor Thyroid Stimulating Thyroid HormoneChatterjee et al., 1989 Hormone α Gene

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

In certain embodiments of the invention, the cells containing nucleicacid constructs of the present invention may be identified in vitro orin vivo by including a marker in the expression construct. Such markerswould confer an identifiable change to the cell permitting easyidentification of cells containing the expression construct. Usually theinclusion of a drug selection marker aids in cloning and in theselection of transformants, for example, genes that confer resistance toneomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol areuseful selectable markers. Alternatively, enzymes such as herpes simplexvirus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT)may be employed. Immunologic markers also can be employed. Theselectable marker employed is not believed to be important, so long asit is capable of being expressed simultaneously with the nucleic acidencoding a gene product. Further examples of selectable markers are wellknown to one of skill in the art.

There are a number of ways in which expression vectors may introducedinto cells. In certain embodiments of the invention, the expressionconstruct comprises a virus or engineered construct derived from a viralgenome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden,1986; Temin, 1986).

One of the preferred methods for in vivo delivery involves the use of anadenovirus expression vector. “Adenovirus expression vector” is meant toinclude those constructs containing adenovirus sequences sufficient to(a) support packaging of the construct and (b) to express apolynucleotide that has been cloned therein. The expression vectorcomprises a genetically engineered form of adenovirus. Knowledge of thegenetic organization of adenovirus, a 36 kB, linear, double-stranded DNAvirus, allows substitution of large pieces of adenoviral DNA withforeign sequences up to 7 kB (Grunhaus and Horwitz, 1992). In contrastto retrovirus, the adenoviral infection of host cells does not result inchromosomal integration because adenoviral DNA can replicate in anepisomal manner without potential genotoxicity. Also, adenoviruses arestructurally stable, and no genome rearrangement has been detected afterextensive amplification. Adenovirus can infect virtually all epithelialcells regardless of their cell cycle stage.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to theinvention. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors, asdescribed by Karlsson et al. (1986), or in the E4 region where a helpercell line or helper virus complements the E4 defect.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1991). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

Retroviral vectors are also suitable for expressing the polynucleotidesof the invention in cells. The retroviruses are a group ofsingle-stranded RNA viruses characterized by an ability to convert theirRNA to double-stranded DNA in infected cells by a process ofreverse-transcription (Coffin, 1990). The resulting DNA then stablyintegrates into cellular chromosomes as a provirus and directs synthesisof viral proteins. The integration results in the retention of the viralgene sequences in the recipient cell and its descendants. The retroviralgenome contains three genes, gag, pol, and env that code for capsidproteins, polymerase enzyme, and envelope components, respectively. Asequence found upstream from the gag gene contains a signal forpackaging of the genome into virions. Two long terminal repeat (LTR)sequences are present at the 5′ and 3′ ends of the viral genome. Thesecontain strong promoter and enhancer sequences and are also required forintegration in the host cell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubinstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988)adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesviruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

In order to affect expression of sense or antisense gene constructs, theexpression construct must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transformingcells lines, or in vivo or ex vivo, as in the treatment of certaindisease states. One mechanism for delivery is via viral infection wherethe expression construct is encapsidated in an infectious viralparticle.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979) andlipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987),gene bombardment using high velocity microprojectiles (Yang et al.,1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu,1988). Some of these techniques may be successfully adapted for in vivoor ex vivo use.

Once the expression construct has been delivered into the cell thenucleic acid encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidencoding the gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

In yet another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. Dubensky et al. (1984) successfully injectedpolyomavirus DNA in the form of calcium phosphate precipitates intoliver and spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty and Neshif (1986) alsodemonstrated that direct intraperitoneal injection of calciumphosphate-precipitated plasmids results in expression of the transfectedgenes. It is envisioned that DNA encoding a gene of interest may also betransferred in a similar manner in vivo and express the gene product.

In still another embodiment of the invention for transferring a nakedDNA expression construct into cells may involve particle bombardment.This method depends on the ability to accelerate DNA-coatedmicroprojectiles to a high velocity allowing them to pierce cellmembranes and enter cells without killing them (Klein et al., 1987).Several devices for accelerating small particles have been developed.One such device relies on a high voltage discharge to generate anelectrical current, which in turn provides the motive force (Yang etal., 1990). The microprojectiles used have consisted of biologicallyinert substances such as tungsten or gold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e., ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentinvention.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Wong et al., (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells. Nicolau et al.,(1987) accomplished successful liposome-mediated gene transfer in ratsafter intravenous injection.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

Other expression constructs which can be employed to deliver a nucleicacid encoding a particular gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide,a galactose-terminal asialganglioside, incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes.Thus, it is feasible that a nucleic acid encoding a particular gene alsomay be specifically delivered into a cell type by any number ofreceptor-ligand systems with or without liposomes. For example,epidermal growth factor (EGF) may be used as the receptor for mediateddelivery of a nucleic acid into cells that exhibit upregulation of EGFreceptor. Mannose can be used to target the mannose receptor on livercells. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T-cellleukemia) and MAA (melanoma) can similarly be used as targetingmoieties.

In a particular example, the oligonucleotide may be administered incombination with a cationic lipid. Examples of cationic lipids include,but are not limited to, lipofectin, DOTMA, DOPE, and DOTAP. Thepublication of WO/0071096, which is specifically incorporated byreference, describes different formulations, such as a DOTAP:cholesterolor cholesterol derivative formulation that can effectively be used forgene therapy. Other disclosures also discuss different lipid orliposomal formulations including nanoparticles and methods ofadministration; these include, but are not limited to, U.S. PatentPublication 20030203865, 20020150626, 20030032615, and 20040048787,which are specifically incorporated by reference to the extent theydisclose formulations and other related aspects of administration anddelivery of nucleic acids. Methods used for forming particles are alsodisclosed in U.S. Pat. Nos. 5,844,107, 5,877,302, 6,008,336, 6,077,835,5,972,901, 6,200,801, and 5,972,900, which are incorporated by referencefor those aspects.

In certain embodiments, gene transfer may more easily be performed underex vivo conditions. Ex vivo gene therapy refers to the isolation ofcells from an animal, the delivery of a nucleic acid into the cells invitro, and then the return of the modified cells back into an animal.This may involve the surgical removal of tissue/organs from an animal orthe primary culture of cells and tissues.

In some embodiments of the invention, it is desirable to inhibit theexpression or activity of miR-29a-c to increase collagen deposition. Forexample, in one embodiment, the invention provides a method of inducingcollagen deposition in a tissue comprising contacting said tissue withan antagonist of miR-29a-c. The antagonist or inhibitor of miR-29a-cfunction may be directed at miR-29a, miR-29b and/or at miR-29c.

The function of miRNAs may be inhibited by the administration ofantagomirs. Initially described by Krützfeldt and colleagues (Krützfeldtet al., 2005), “antagomirs” are single-stranded, chemically-modifiedribonucleotides that are at least partially complementary to the miRNAsequence. Antagomirs may comprise one or more modified nucleotides, suchas 2′-O-methyl-sugar modifications. In some embodiments, antagomirscomprise only modified nucleotides. Antagomirs may also comprise one ormore phosphorothioate linkages resulting in a partial or fullphosphorothioate backbone. To facilitate in vivo delivery and stability,the antagomir may be linked to a cholesterol moiety at its 3′ end.Antagomirs suitable for inhibiting miRNAs may be about 15 to about 50nucleotides in length, more preferably about 18 to about 30 nucleotidesin length, and most preferably about 20 to about 25 nucleotides inlength. “Partially complementary” refers to a sequence that is at leastabout 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to atarget polynucleotide sequence. The antagomirs may be at least about75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to a maturemiRNA sequence. In some embodiments, the antagomir may be substantiallycomplementary to a mature miRNA sequence, that is at least about 95%,96%, 97%, 98%, or 99% complementary to a target polynucleotide sequence.In other embodiments, the antagomirs are 100% complementary to themature miRNA sequence.

In one embodiment, the antagonist of miR-29a-c is an antagomir. Theantagomir may comprise a sequence that is at least partiallycomplementary to the mature miRNA sequence of miR-29a, miR-29b, ormiR-29c. In another embodiment, the antagomir comprises a sequence thatis at least partially complementary to the sequence of SEQ ID NO: 18,SEQ ID NO: 19, or SEQ ID NO: 20. In another embodiment, the antagomircomprises a sequence that is 100% complementary to SEQ ID NO: 18, SEQ IDNO: 19, or SEQ ID NO: 20.

Inhibition of microRNA function may also be achieved by administeringantisense oligonucleotides targeting the mature miR-29a, miR-29b ormiR-29c sequences. The antisense oligonucleotides may be ribonucleotidesor deoxyribonucleotides. Preferably, the antisense oligonucleotides haveat least one chemical modification. Antisense oligonucleotides may becomprised of one or more “locked nucleic acids”. “Locked nucleic acids”(LNAs) are modified ribonucleotides that contain an extra bridge betweenthe 2′ and 4′ carbons of the ribose sugar moiety resulting in a “locked”conformation that confers enhanced thermal stability to oligonucleotidescontaining the LNAs. Alternatively, the antisense oligonucleotides maycomprise peptide nucleic acids (PNAs), which contain a peptide-basedbackbone rather than a sugar-phosphate backbone. Other chemicalmodifications that the antisense oligonucleotides may contain include,but are not limited to, sugar modifications, such as 2′-O-alkyl (e.g.2′-O-methyl, 2′-O-methoxyethyl), 2′-fluoro, and 4′ thio modifications,and backbone modifications, such as one or more phosphorothioate,morpholino, or phosphonocarboxylate linkages (see, for example, U.S.Pat. Nos. 6,693,187 and 7,067,641, which are herein incorporated byreference in their entireties). In some embodiments, suitable antisenseoligonucleotides are 2′-O-methoxyethyl “gapmers” which contain2′-O-methoxyethyl-modified ribonucleotides on both 5′ and 3′ ends withat least ten deoxyribonucleotides in the center. These “gapmers” arecapable of triggering RNase H-dependent degradation mechanisms of RNAtargets. Other modifications of antisense oligonucleotides to enhancestability and improve efficacy, such as those described in U.S. Pat. No.6,838,283, which is herein incorporated by reference in its entirety,are known in the art and are suitable for use in the methods of theinvention. Preferable antisense oligonucleotides useful for inhibitingthe activity of microRNAs are about 19 to about 25 nucleotides inlength. Antisense oligonucleotides may comprise a sequence that is atleast partially complementary to a mature miRNA sequence, e.g. at leastabout 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to amature miRNA sequence. In some embodiments, the antisenseoligonucleotide may be substantially complementary to a mature miRNAsequence, that is at least about 95%, 96%, 97%, 98%, or 99%complementary to a target polynucleotide sequence. In one embodiment,the antisense oligonucleotide comprises a sequence that is 100%complementary to a mature miRNA sequence.

In another embodiment of the invention, the antagonist of miR-29a-c is achemically-modified antisense oligonucleotide. The chemically-modifiedantisense oligonucleotide may comprise a sequence that is at leastpartially complementary to the mature miRNA sequence of miR-29a,miR-29b, or miR-29c. In yet another embodiment, the chemically-modifiedantisense oligonucleotide comprises a sequence that is at leastpartially complementary to the sequence of SEQ ID NO: 18, SEQ ID NO: 19,or SEQ ID NO: 20. In another embodiment, the chemically-modifiedantisense oligonucleotide comprises a sequence that is 100%complementary to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

Antisense oligonucleotides may comprise a sequence that is substantiallycomplementary to a precursor miRNA sequence (pre-miRNA) for miR-29a-c.In some embodiments, the antisense oligonucleotide comprises a sequencethat is substantially complementary to a sequence located outside thestem-loop region of the pre-miR-29a, pre-miR-29b, or pre-miR-29csequence.

Another approach for inhibiting the function of miR-29a-c isadministering an inhibitory RNA molecule having at least partialsequence identity to the mature miR-29a, miR-29b and miR-29c sequences.The inhibitory RNA molecule may be a double-stranded, small interferingRNA (siRNA) or a short hairpin RNA molecule (shRNA) comprising astem-loop structure. The double-stranded regions of the inhibitory RNAmolecule may comprise a sequence that is at least partially identical,e.g. about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical, tothe mature miRNA sequence. In some embodiments, the double-strandedregions of the inhibitory RNA comprise a sequence that is at leastsubstantially identical to the mature miRNA sequence. “Substantiallyidentical” refers to a sequence that is about 95%, 96%, 97%, 98%, or 99%identical to a target polynucleotide sequence. In other embodiments, thedouble-stranded regions of the inhibitory RNA molecule may be 100%identical to the target miRNA sequence.

In one embodiment, an antagonist of miR-29a-c is an inhibitory RNAmolecule comprising a double-stranded region, wherein thedouble-stranded region comprises a sequence having 100% identity to themature miR-29a (SEQ ID NO: 18), miR-29b (SEQ ID NO: 19), or miR-29c (SEQID NO: 20) sequence. In some embodiments, antagonists of miR-29a-c areinhibitory RNA molecules which comprise a double-stranded region,wherein said double-stranded region comprises a sequence of at leastabout 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to themature miR-29a, miR-29b, or miR-29c sequence.

In another embodiment, an inhibitory RNA molecule may be a ribozyme. Aribozyme is a catalytic RNA that hydrolyzes phosphodiester bonds of RNAmolecules. The ribozyme may be designed to target one or more ofmiR-29a, miR-29b, and miR-29c resulting in their hydrolysis.

In certain embodiments, expression vectors are employed to express anantagonist of miR-29a-c (e.g., antagomirs, antisense oligonucleotides,and inhibitory RNA molecules). In one embodiment, an expression vectorfor expressing an antagonist of miR-29a-c comprises a promoter operablylinked to a polynucleotide encoding an antisense oligonucleotide,wherein the sequence of the expressed antisense oligonucleotide is atleast partially complementary to the mature miR-29a, miR-29b, or miR-29csequence. In yet another embodiment, an expression vector for expressingan inhibitor of miR-29a-c comprises one or more promoters operablylinked to a polynucleotide encoding a shRNA or siRNA, wherein theexpressed shRNA or siRNA comprises a sequence that is identical,partially identical, or substantially identical to the mature miR-29a,miR-29b, or miR-29c sequence. “Partially identical” refers to a sequencethat is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to a target polynucleotide sequence. “Substantially identical”refers to a sequence that is at least about 95%, 96%, 97%, 98%, or 99%identical to a target polynucleotide sequence.

Current medical management of cardiac hypertrophy in the setting of acardiovascular disorder includes the use of at least two types of drugsinhibitors of the renin-angiotensin system, and β-adrenergic blockingagents (Bristow, 1999). Therapeutic agents to treat pathologichypertrophy in the setting of heart failure include angiotensin IIconverting enzyme (ACE) inhibitors and β-adrenergic receptor blockingagents (Eichhorn and Bristow, 1996). Other pharmaceutical agents thathave been disclosed for treatment of cardiac hypertrophy includeangiotensin II receptor antagonists (U.S. Pat. No. 5,604,251) andneuropeptide Y antagonists (WO 98/33791). Despite currently availablepharmaceutical compounds, prevention and treatment of cardiachypertrophy, and subsequent heart failure, continue to present atherapeutic challenge.

Non-pharmacological treatment is primarily used as an adjunct topharmacological treatment. One means of non-pharmacological treatmentinvolves reducing the sodium in the diet. In addition,non-pharmacological treatment also entails the elimination of certainprecipitating drugs, including negative inotropic agents (e.g., certaincalcium channel blockers and antiarrhythmic drugs like disopyramide),cardiotoxins (e.g., amphetamines), and plasma volume expanders (e.g.,nonsteroidal anti-inflammatory agents and glucocorticoids).

The present invention provides a method of treating cardiac fibrosis,cardiac hypertrophy or heart failure in a subject in need thereofcomprising identifying a subject having cardiac fibrosis, cardiachypertrophy or heart failure; and administering to the subject anagonist of miR-29 expression or function. Preferably, administration ofa miR-29 agonist results in the improvement of one or more symptoms ofpathologic cardiac fibrosis, hypertrophy or heart failure in thesubject, or in the delay in the transition from cardiac hypertrophy toheart failure. The one or more improved symptoms may be increasedexercise capacity, increased cardiac ejection volume, decreased leftventricular end diastolic pressure, decreased pulmonary capillary wedgepressure, increased cardiac output, or cardiac index, lowered pulmonaryartery pressures, decreased left ventricular end systolic and diastolicdimensions, decreased cardiac fibrosis, decreased collagen deposition incardiac muscle, decreased left and right ventricular wall stress,decreased wall tension, increased quality of life, and decreased diseaserelated morbidity or mortality. In addition, use of agonists ofmiR-29a-c may prevent cardiac hypertrophy and its associated symptomsfrom arising either directly or indirectly.

In another embodiment, there is provided a method of preventingpathologic hypertrophy or heart failure in a subject in need thereofcomprising identifying a subject at risk of developing pathologiccardiac hypertrophy or heart failure; and promoting the expression oractivity of miR-29a-c in cardiac cells of the subject. Cardiac cellsinclude cardiac myocytes, fibroblasts, smooth muscle cells, endothelialcells, and any other cell type normally found in cardiac tissue. ThemiR-29a-c agonist may be an agonist of miR-29a, miR-29b and/or miR-29c.The subject at risk may exhibit one or more of a list of risk factorscomprising cardiac fibrosis, low expression of miR-29, long-standinguncontrolled hypertension, uncorrected valvular disease, chronic angina,recent myocardial infarction, congenital predisposition to heart diseaseand/or pathological hypertrophy, and/or may be diagnosed as having agenetic predisposition to cardiac hypertrophy, and/or may have afamilial history of cardiac hypertrophy.

In another embodiment, there is provided a method of treating myocardialinfarction in a subject in need thereof comprising promoting expressionor activity of miR-29a-c in cardiac cells of the subject. In anotherembodiment, the invention provides a method of preventing cardiachypertrophy and dilated cardiomyopathy in a subject in need thereofcomprising promoting expression or activity of miR-29a-c in cardiaccells of the subject. In another embodiment, the invention provides amethod of inhibiting progression of cardiac hypertrophy in a subject inneed thereof comprising promoting expression or activity of miR-29a-c incardiac cells of the subject. Another embodiment is a method ofincreasing exercise tolerance in a subject with heart failure or cardiachypertrophy comprising promoting expression or activity of miR-29a-c incardiac cells of the subject. Another embodiment is a method of reducinghospitalization in a subject with heart failure or cardiac hypertrophycomprising promoting expression or activity of miR-29a-c in cardiaccells of the subject. In some embodiments, the invention providesmethods for improving quality of life and decreasing morbidity ormortality in a subject with heart failure or cardiac hypertrophycomprising promoting expression or activity of miR-29a-c in cardiaccells of the subject.

Treatment regimens would vary depending on the clinical situation.However, long-term maintenance would appear to be appropriate in mostcircumstances. It also may be desirable to treat hypertrophy withagonists of miR-29a-c intermittently, such as within a brief windowduring disease progression.

In addition, the miR-29 family is involved in the regulation of cardiacfibrosis. Since this miR family is enriched in fibroblasts compared tomyocytes, it is likely that myocytes secrete a factor, possibly BNP,which upregulates the miR-29 family in fibroblast cells and thusprotects against the development of cardiac fibrosis. This factor isvery high in miR-208 KO mice, which correlates with the upregulation ofmiR-29a-c and repression of fibrosis. miR-29a-c levels are elevated innormal heart disease, which is likey a protective effect to limitcollagen deposition. Thus, the particular use of miR-29a-c and agoniststhereof in repressing cardiac fibrosis and collagen deposition incardiac tissues is contemplated. A comparable mechanism for theactivation of the miR-29 family may be applicable in skeletal musclefibrosis as well. miR-29a-c regulates the expression of a number ofextracellular matrix genes, such as fibrillin 1 (FBN1), collagen type I,α1 (COL1A1), collagen type I α2 (COL1A2), and collagen type III, α1(COL3A1) (see Example 4). Accordingly, the present invention alsoprovides methods of regulating one or more extracellular matrix genes ina cell.

In one embodiment, the method comprises contacting the cell with anagonist of miR-29 a-c. In another embodiment, the method comprisescontacting the cell with an antagonist of miR-29a-c. In still anotherembodiment, the one or more extracellular matrix genes include fibrillin1 (FBN1), collagen type I, α1 (COL1A1), collagen type I α2 (COL1A2), andcollagen type III, α1 (COL3A1). In some embodiments, the one or moreextracellular matrix genes are upregulated following contact of the cellwith an antagonist of miR-29a-c. In other embodiments, the one or moreextracellular matrix genes are downregulated following contact of thecell with an agonist of miR-29a-c.

The inventors have demonstrated that miR-29a-c expression was decreasedin cardiac fibroblasts exposed to TGFβ, suggesting that the decrease inmiR-29a-c following myocardial infarction might be TGFβ-regulated(Example 5). Interestingly, natriuretic peptides like B-type natriureticpeptide (BNP) have been shown to inhibit TGFβ-regulated gene expressionrelated to fibrosis and myofibroblast conversion (Kapoun et al., 2004).In this regard, the inventors reported previously that mice lacking thecardiac-specific miRNA miR-208 were resistant to cardiac fibrosis andremodeling and exhibited increased expression of BNP at baseline (vanRooij et al., 2007). Since BNP is known to antagonize the effects ofTGFβ, the inventors suggest that the increased levels of BNP in thesemice might enhance the expression of miR-29a-c. Indeed, a dose-dependentincrease in miR-29a-c expression was observed upon removal of miR-208,which coincided with an increasing expression level of BNP (Example 5).These data indicate that TGFβ induces the expression of collagen relatedgenes in fibroblasts at least partly through decreasing the level ofmiR-29a-c, which can be inhibited by BNP secreted by cardiomyocytes.Thus, the present invention provides a method of increasing miR-29a-cexpression and/or activity in a subject by administering at least oneTGFβ inhibitor. TGFβ inhibitors may include anti-TGFβ antibodies, TGFβantisense molecules, and small molecules that inhibit TGFβ activity asdescribed in U.S. Pat. No. 6,509,318, which is herein incorporated byreference in its entirety. TGFβ inhibitors may also be used inconjuction with miR-29a-c agonists as a combination therapy to treatcardiac fibrosis, cardiac hypertrophy, or heart failure in a subject.TGFβ inhibitors may also be co-administered with miR-29a-c agonists totreat or prevent tissue fibrosis in a subject.

In addition to playing an important role in controlling fibrosis in theheart, the ubiquitous expression of the miR-29 family suggests that italso may play a role in other fibrotic indications, such as thoseinvolving the kidney, liver and lungs. Fibrosis is also observedsecondary to diabetes. Type 1 and type 2 diabetic patients are atincreased risk of cardiomyopathy. Cardiomyopathy in diabetes isassociated with a cluster of features, including decreased diastoliccompliance, interstitial fibrosis, and myocyte hypertrophy.

The present invention also provides a method of treating or preventing atissue fibrosis in a subject. In one embodiment, the method comprisesidentifying a subject having or at risk of tissue fibrosis; andincreasing the expression and/or activity of miR-29a-c in skeletalmuscle or fibroblast cells of the subject. In another embodiment, thetissue fibrosis is cardiac fibrosis, scleroderma (localized orsystemic), skeletal muscle fibrosis, hepatic fibrosis, kidney fibrosis,pulmonary fibrosis, or diabetic fibrosis. In some embodiments,increasing the expression and/or activity of miR-29a-c comprisesadministering an agonist of miR-29a-c to the subject. In otherembodiments, increasing the expression and/or activity of miR-29a-ccomprises administering to the subject an expression vector that encodesmiR-29a-c. In another embodiment, the method further comprisesadministering to the subject a non-miR-29a-c fibrotic therapy.

The present invention encompasses methods of treating tissue fibrosisassociated with one or more conditions or disorders in a subject in needthereof. In one embodiment, the method comprises administering to thesubject an agonist of miR-29a-c. In another embodiment, the methodcomprises administering to the subject an expression vector that encodesmiR-29a-c. The one or more conditions or disorders associated withtissue fibrosis may include, but are not limited to, congenital hepaticfibrosis (CHF); renal tubulointerstitial fibrosis; pulmonary fibrosisassociated with an autoimmune disorder (e.g. rheumatoid arthritis, lupusand sarcoidosis); interstitial fibrosis associated with diabeticcardiomyopathy; skeletal muscle fibrosis associated with musculardystrophies (e.g. Becker muscular dystrophy and Duchenne musculardystrophy), denervation atrophies, and neuromuscular diseases (e.g.acute polyneuritis, poliomyelitis, Werdig/Hoffman disease, amyotrophiclateral sclerosis, and progressive bulbar atrophy disease).

The present invention also contemplates methods of treatingpathologies/deficiencies that are characterized by the loss, lack, orunderproduction of collagen. Using an antagonist of miR-29a-c, theexpression of collagen can be increased to replace missing collagen orsupplement existing collagen where there is a need. Thus, the presentinvention provides a method of inducing collagen deposition in a tissuecomprising contacting said tissue with an antagonist of miR-29a-c. Theantagonist may be directed at miR-29a, miR-29b and/or at miR-29c. In oneembodiment, the antagonist comprises a sequence that is complementary toSEQ ID NO: 18. In another embodiment, the antagonist comprises asequence that is complementary to SEQ ID NO: 19. In another embodiment,the antagonist comprises a sequence that is complementary to SEQ ID NO:20. The antagonist may be an antagomir of miR-29a-c, an antisenseoligonucleotide that targets a mature miR-29a-c sequence, or aninhibitory RNA molecule, such as a siRNA or a shRNA, that comprises asequence identical to a mature miR-29a-c sequence, a ribozyme or anyother inhibitory nucleic acid. The antagonist may be linked orconjugated to agents that facilitate the entry of the antagonist intocells or tissues. Various conditions and disorders in which an increasein collagen deposition would be beneficial and can be treated byadministering an antagonist of miR-29a-c include, but are not limitedto, Ehlers-Danlos syndrome (EDS); Vitamin C deficiency (a.k.a scurvy);aging of the skin (e.g. natural aging and photoaging due to sun damage);and stretch marks (striae).

Ehlers-Danlos syndrome (EDS) is a group of rare genetic disordersaffecting humans and domestic animals caused by a defect in collagensynthesis. Depending on the individual mutation, the severity of thedisease can vary from mild to life-threatening. Mutations in theADAMTS2, COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, PLOD1 and TNXB genescause EDS. Mutations in these genes usually alter the structure,production, or processing of collagen or proteins that interact withcollagen. A defect in collagen can weaken connective tissue in the skin,bones, blood vessels, and organs, resulting in the features of thedisorder. Thus, collagen deposition induced by miR-29a-c antagonists ofthe invention would act to replenish the level of normal collagen in EDSpatients and alleviate symptoms of the disease. Similarly,administration of an antagonist of miR-29a-c would benefit subjectssuffering from vitamin C deficiency or scurvy. Vitamin C deficiency is adisease that results from insufficient intake of vitamin C, which isrequired for normal collagen synthesis in humans.

Collagen deposition in tissues resulting from the administration of anantagonist of miR-29a-c would also be useful in various cosmeticapplications. Effects of aging of the skin produced by natural agingprocesses or photodamage resulting from over-exposure to the sun couldbe reduced by administering to a subject in need thereof a miR-29a-cantagonist. Administration of miR-29a-c antagonists may also facilitatethe disappearance of stretch marks. Stretch marks are a form of scarringon the skin that are caused by tearing of the dermis. Stretch marks arethe result of the rapid stretching of the skin associated with rapidgrowth (common in puberty) or weight gain (e.g., pregnancy).

The tissue to which the inventive methods may be applied include facialtissue, such a forehead tissue, a lip, a cheek, a chin, an eyebrow, aneyelid, under the eye, or near the mouth, hand tissue, neck tissue, armtissue, leg tissue, stomach tissue or breast tissue. In someembodiments, the tissue may comprise a wound, a skin graft, scar tissue,wrinkles, lax skin, sun damage, chemical damage, heat damage, colddamage, and/or stretch marks.

In another embodiment of the invention, the contacting of the tissuewith the miR-29a-c antagonist comprises injection into said tissue,injection into vasculature that feeds said tissue, or topicalapplication. The topical application may be an ointment, cream, gel,salve, or balm. In another embodiment, the method further comprises useof a pressure bandage or dressing. The antagonist of miR-29a-c may becontacted with said tissue more than once. In some embodiments, theantagonist is contacted with said tissue 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 times. In otherembodiments, the antagonist is contacted with said tissue over 2, 3, 4,5, or 6 days, 1, 2, 3, or 4 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 11months, or 1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 years.

In still another embodiment, the method further comprises contactingsaid tissue with a second agent. The second agent may include, but isnot limited to, topical vitamin A, topical vitamin C, or vitamin E. Inanother embodiment, the method further comprises subjecting said tissueto a second treatment. The second treatment may comprise a chemicalpeel, laser treatment, dermaplaning, or dermabrasion. In anotherembodiment, the tissue is in a subject that suffers from Ehler's-Danlossyndrome or Vitamin C deficiency.

The present invention also contemplates the use of miR-29a-c antagonistsas profibrotic agents to convert soft plaques in the vasculature intofibrotic tissue to prevent myocardial infarction. Soft plaques are abuild-up of lipids containing predominantly cholesterol that lieunderneath the endothelial lining of the arterial wall. Recently, it wasrecognized that these soft plaques are prone to rupture resulting in theformation of a blood clot, which can potentially block blood flowthrough the artery and cause a heart attack (i.e. myocardialinfarction). It is these soft plaques that are often responsible incausing a healthy subject with no symptoms to suffer a seeminglyunexpected heart attack. After a soft plaque ruptures, the vessel wallheals and the soft plaque becomes a hard plaque, which rarely causefurther problems. Thus, strategies for converting soft plaques intofibrotic tissue would prevent the soft plaques from rupturing andpossibly inducing a myocardial infarction.

As described in detail above, inhibition of miR-29a-c leads to anincrease in collagen deposition and the formation of fibrotic tissue.Accordingly, the present invention provides a method for increasingfibrotic tissue formation in the wall of a vessel comprising deliveringan antagonist of miR-29a-c to one or more soft plaque sites in thevessel wall, wherein the soft plaque is converted to fibrotic tissuefollowing delivery of the antagonist of miR-29a-c. Soft plaques can beidentified by methods known in the art, including, but not limited to,intravascular ultrasound and computed tomography (Sahara et al. (2004)European Heart Journal, Vol. 25: 2026-2033; Budhoff (2006) J. Am. Coll.Cardiol., Vol. 48: 319-321; Hausleiter et al. (2006) J. Am. Coll.Cardiol., Vol. 48: 312-318). Any of the miR-29a-c antagonists describedherein are suitable for use in the method.

The miR-29a-c antagonist may delivered to the one or more soft plaquesites by direct injection or by using a catheter or a device thatisolates the coronary circulation. In one embodiment, the miR-29a-cantagonist is delivered to the one or more soft plaque sites by amedical device used in vascular surgery, such as a stent or balloon. ThemiR-29 antagonist may be coated on a metal stent to form a drug-elutingstent. A drug-eluting stent is a scaffold that holds open narrowed ordiseased arteries and releases a compound to prevent cellularproliferation and/or inflammation. miR-29a-c antagonists may be appliedto a metal stent imbedded in a thin polymer for release of the miR-29a-cover time. Methods of coating stents with therapeutic compounds areknown in the art. See, e.g., U.S. Pat. No. 7,144,422; U.S. Pat. No.7,055,237; and WO 2004/004602, which are here incorporated by referencein their entireties. In some embodiments, the miR-29a-c may be used incombination with other anti-restenosis compounds to produce aformulation for incorporation into drug-eluting stents and balloons.Suitable compounds for use in combination with the antagonists ofmiR-29a-c include, but are not limited to, paclitaxel, rapamycin(sirolimus), tacrolimus, zotarolimus, everolimus, docetaxel,pimecrolimus, and derivatives thereof.

The present invention also contemplates methods for scavenging orclearing a miR-29a-c agonist following treatment. In one embodiment, themethod comprises overexpression of binding site regions for miR-29a-c infibroblasts using a fibroblast specific promoter. The binding siteregions preferably contain a sequence of the seed region for miR-29a-c.In some embodiments, the binding site may contain a sequence from the3′UTR of one or more targets of miR-29a-c, such as COL1A1, COL1A2,COL1A3 and/or FBN1. In another embodiment, a miR-29a-c antagonist may beadministered after a miR-29a-c agonist to attenuate or stop the functionof the microRNA. In another embodiment, the present invention provides amethod for scavenging or clearing miR-29a-c antagonists followingtreatment. The method may comprise overexpressing binding sites for themiR-20a-c antagonists in fibroblasts or other tissue in which amiR-29a-c antagonist was administered.

Combined Therapy

In another embodiment, it is envisioned to use an agonist of miR-29a-cin combination with other therapeutic modalities for treating cardiachypertrophy, heart failure and myocardial infarction. Thus, one may alsoprovide to the subject more “standard” pharmaceutical cardiac therapiesin combination with the miR-29a-c agonist. Examples of other therapiesinclude, without limitation, so-called “beta blockers,”anti-hypertensives, cardiotonics, anti-thrombotics, vasodilators,hormone antagonists, iontropes, diuretics, endothelin receptorantagonists, calcium channel blockers, phosphodiesterase inhibitors, ACEinhibitors, angiotensin type 2 antagonists and cytokineblockers/inhibitors, and HDAC inhibitors.

Combinations may be achieved by contacting cardiac cells with a singlecomposition or pharmacological formulation that includes an agonist ofmiR-29a-c and a standard pharmaceutical agent, or by contacting the cellwith two distinct compositions or formulations, at the same time,wherein one composition includes an agonist of miR-29a-c and the otherincludes the standard pharmaceutical agent. Alternatively, the therapyusing an agonist of miR-29a-c may precede or follow administration ofthe other agent(s) by intervals ranging from minutes to weeks. Inembodiments where the standard pharmaceutical agent and miR-29a-cagonist are applied separately to the cell, one would generally ensurethat a significant period of time did not expire between the time ofeach delivery, such that the pharmaceutical agent and miR-29a-c agonistwould still be able to exert an advantageously combined effect on thecell. In such instances, it is contemplated that one would typicallycontact the cell with both modalities within about 12-24 hours of eachother and, more preferably, within about 6-12 hours of each other, witha delay time of only about 12 hours being most preferred. In somesituations, it may be desirable to extend the time period for treatmentsignificantly, however, where several days (2, 3, 4, 5, 6 or 7) toseveral weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

It also is conceivable that more than one administration of either anagonist of miR-29a-c, or the other pharmaceutical agent will be desired.In this regard, various combinations may be employed. By way ofillustration, where the agonist of miR-29a-c is “A” and the other agentis “B,” the following permutations based on 3 and 4 totaladministrations are exemplary:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A  B/B/A/BA/A/B/B  A/B/A/B  A/B/B/A  B/B/A/A  B/A/B/A  B/A/A/B  B/B/B/AA/A/A/B  B/A/A/A  A/B/A/A  A/A/B/A  A/B/B/B  B/A/B/B  B/B/A/BOther combinations are likewise contemplated.

Pharmacological therapeutic agents and methods of administration,dosages, etc., are well known to those of skill in the art (see forexample, the “Physicians Desk Reference”, Klaassen's “ThePharmacological Basis of Therapeutics”, “Remington's PharmaceuticalSciences”, and “The Merck Index, Eleventh Edition”, incorporated hereinby reference in relevant parts), and may be combined with the inventionin light of the disclosures herein. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject, and suchindividual determinations are within the skill of those of ordinaryskill in the art.

Non-limiting examples of a pharmacological therapeutic agent that may beused in the present invention include an antihyperlipoproteinemic agent,an antiarteriosclerotic agent, an antithrombotic/fibrinolytic agent, ablood coagulant, an antiarrhythmic agent, an antihypertensive agent, avasopressor, a treatment agent for congestive heart failure, anantianginal agent, an antibacterial agent or a combination thereof.

In addition, it should be noted that any of the following may be used todevelop new sets of cardiac therapy target genes as β-blockers were usedin the present examples (see below). While it is expected that many ofthese genes may overlap, new gene targets likely can be developed.

In certain embodiments, administration of an agent that lowers theconcentration of one of more blood lipids and/or lipoproteins, knownherein as an “antihyperlipoproteinemic,” may be combined with acardiovascular therapy according to the present invention, particularlyin treatment of athersclerosis and thickenings or blockages of vasculartissues. In certain embodiments, an antihyperlipoproteinemic agent maycomprise an aryloxyalkanoic/fibric acid derivative, a resin/bile acidsequesterant, a HMG CoA reductase inhibitor, a nicotinic acidderivative, a thyroid hormone or thyroid hormone analog, a miscellaneousagent or a combination thereof.

Non-limiting examples of aryloxyalkanoic/fibric acid derivatives includebeclobrate, enzafibrate, binifibrate, ciprofibrate, clinofibrate,clofibrate (atromide-S), clofibric acid, etofibrate, fenofibrate,gemfibrozil (lobid), nicofibrate, pirifibrate, ronifibrate, simfibrateand theofibrate.

Non-limiting examples of resins/bile acid sequesterants includecholestyramine (cholybar, questran), colestipol (colestid) andpolidexide.

Non-limiting examples of HMG CoA reductase inhibitors include lovastatin(mevacor), pravastatin (pravochol) or simvastatin (zocor).

Non-limiting examples of nicotinic acid derivatives include nicotinate,acepimox, niceritrol, nicoclonate, nicomol and oxiniacic acid.

Non-limiting examples of thyroid hormones and analogs thereof includeetoroxate, thyropropic acid and thyroxine.

Non-limiting examples of miscellaneous antihyperlipoproteinemics includeacifran, azacosterol, benfluorex, β-benzalbutyramide, carnitine,chondroitin sulfate, clomestrone, detaxtran, dextran sulfate sodium, 5,8, 11, 14, 17-eicosapentaenoic acid, eritadenine, furazabol, meglutol,melinamide, mytatrienediol, ornithine, γ-oryzanol, pantethine,pentaerythritol tetraacetate, α-phenylbutyramide, pirozadil, probucol(lorelco), β-sitosterol, sultosilic acid-piperazine salt, tiadenol,triparanol and xenbucin. Non-limiting examples of anantiarteriosclerotic include pyridinol carbamate.

In certain embodiments, administration of an agent that aids in theremoval or prevention of blood clots may be combined with administrationof a modulator, particularly in treatment of athersclerosis andvasculature (e.g., arterial) blockages. Non-limiting examples ofantithrombotic and/or fibrinolytic agents include anticoagulants,anticoagulant antagonists, antiplatelet agents, thrombolytic agents,thrombolytic agent antagonists or combinations thereof.

In certain embodiments, antithrombotic agents that can be administeredorally, such as, for example, aspirin and wafarin (coumadin), arepreferred.

Non-limiting examples of anticoagulants include acenocoumarol, ancrod,anisindione, bromindione, clorindione, coumetarol, cyclocumarol, dextransulfate sodium, dicumarol, diphenadione, ethyl biscoumacetate,ethylidene dicoumarol, fluindione, heparin, hirudin, lyapolate sodium,oxazidione, pentosan polysulfate, phenindione, phenprocoumon, phosvitin,picotamide, tioclomarol and warfarin.

Non-limiting examples of antiplatelet agents include aspirin, a dextran,dipyridamole (persantin), heparin, sulfinpyranone (anturane) andticlopidine (ticlid).

Non-limiting examples of thrombolytic agents include tissue plaminogenactivator (activase), plasmin, pro-urokinase, urokinase (abbokinase)streptokinase (streptase), anistreplase/APSAC (eminase).

In certain embodiments wherein a subject is suffering from a hemorrhageor an increased likelihood of hemorrhaging, an agent that may enhanceblood coagulation may be used. Non-limiting examples of a bloodcoagulation promoting agents include thrombolytic agent antagonists andanticoagulant antagonists.

Non-limiting examples of anticoagulant antagonists include protamine andvitamine K1.

Non-limiting examples of thrombolytic agent antagonists includeamiocaproic acid (amicar) and tranexamic acid (amstat). Non-limitingexamples of antithrombotics include anagrelide, argatroban, cilstazol,daltroban, defibrotide, enoxaparin, fraxiparine, indobufen, lamoparan,ozagrel, picotamide, plafibride, tedelparin, ticlopidine and triflusal.

Non-limiting examples of antiarrhythmic agents include Class Iantiarrhythmic agents (sodium channel blockers), Class II antiarrhythmicagents (beta-adrenergic blockers), Class III antiarrhythmic agents(repolarization prolonging drugs), Class IV antiarrhythmic agents(calcium channel blockers) and miscellaneous antiarrhythmic agents.

Non-limiting examples of sodium channel blockers include Class IA, ClassIB and Class IC antiarrhythmic agents. Non-limiting examples of Class IAantiarrhythmic agents include disppyramide (norpace), procainamide(pronestyl) and quinidine (quinidex). Non-limiting examples of Class IBantiarrhythmic agents include lidocaine (xylocalne), tocainide(tonocard) and mexiletine (mexitil). Non-limiting examples of Class ICantiarrhythmic agents include encainide (enkaid) and flecainide(tambocor).

Non-limiting examples of a β-blocker, otherwise known as a β-adrenergicblocker, a β-adrenergic antagonist or a Class II antiarrhythmic agent,include acebutolol (sectral), alprenolol, amosulalol, arotinolol,atenolol, befunolol, betaxolol, bevantolol, bisoprolol, bopindolol,bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol, butidrinehydrochloride, butofilolol, carazolol, carteolol, carvedilol,celiprolol, cetamolol, cloranolol, dilevalol, epanolol, esmolol(brevibloc), indenolol, labetalol, levobunolol, mepindolol,metipranolol, metoprolol, moprolol, nadolol, nadoxolol, nifenalol,nipradilol, oxprenolol, penbutolol, pindolol, practolol, pronethalol,propanolol (inderal), sotalol (betapace), sulfinalol, talinolol,tertatolol, timolol, toliprolol and xibinolol. In certain embodiments,the beta blocker comprises an aryloxypropanolamine derivative.Non-limiting examples of aryloxypropanolamine derivatives includeacebutolol, alprenolol, arotinolol, atenolol, betaxolol, bevantolol,bisoprolol, bopindolol, bunitrolol, butofilolol, carazolol, carteolol,carvedilol, celiprolol, cetamolol, epanolol, indenolol, mepindolol,metipranolol, metoprolol, moprolol, nadolol, nipradilol, oxprenolol,penbutolol, pindolol, propanolol, talinolol, tertatolol, timolol andtoliprolol.

Non-limiting examples of an agent that prolong repolarization, alsoknown as a Class III antiarrhythmic agent, include amiodarone(cordarone) and sotalol (betapace).

Non-limiting examples of a calcium channel blocker, otherwise known as aClass IV antiarrhythmic agent, include an arylalkylamine (e.g.,bepridile, diltiazem, fendiline, gallopamil, prenylamine, terodiline,verapamil), a dihydropyridine derivative (felodipine, isradipine,nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine) apiperazinde derivative (e.g., cinnarizine, flunarizine, lidoflazine) ora miscellaneous calcium channel blocker such as bencyclane, etafenone,magnesium, mibefradil or perhexyline. In certain embodiments a calciumchannel blocker comprises a long-acting dihydropyridine(nifedipine-type) calcium antagonist.

Non-limiting examples of miscellaneous antiarrhythmic agents includeadenosine (adenocard), digoxin (lanoxin), acecainide, ajmaline,amoproxan, aprindine, bretylium tosylate, bunaftine, butobendine,capobenic acid, cifenline, disopyranide, hydroquinidine, indecainide,ipatropium bromide, lidocaine, lorajmine, lorcainide, meobentine,moricizine, pirmenol, prajmaline, propafenone, pyrinoline, quinidinepolygalacturonate, quinidine sulfate and viquidil.

Non-limiting examples of antihypertensive agents include sympatholytic,alpha/beta blockers, alpha blockers, anti-angiotensin II agents, betablockers, calcium channel blockers, vasodilators and miscellaneousantihypertensives.

Non-limiting examples of an α-blocker, also known as an α-adrenergicblocker or an α-adrenergic antagonist, include amosulalol, arotinolol,dapiprazole, doxazosin, ergoloid mesylates, fenspiride, indoramin,labetalol, nicergoline, prazosin, terazosin, tolazoline, trimazosin andyohimbine. In certain embodiments, an alpha blocker may comprise aquinazoline derivative. Non-limiting examples of quinazoline derivativesinclude alfuzosin, bunazosin, doxazosin, prazosin, terazosin andtrimazosin.

In certain embodiments, an antihypertensive agent is both an α- andβ-adrenergic antagonist. Non-limiting examples of an alpha/beta blockercomprise labetalol (normodyne, trandate).

Non-limiting examples of anti-angiotensin II agents include angiotensinconverting enzyme inhibitors and angiotensin II receptor antagonists.Non-limiting examples of angiotensin converting enzyme inhibitors (ACEinhibitors) include alacepril, enalapril (vasotec), captopril,cilazapril, delapril, enalaprilat, fosinopril, lisinopril, moveltopril,perindopril, quinapril and ramipril. Non-limiting examples of anangiotensin II receptor blocker, also known as an angiotensin IIreceptor antagonist, an ANG receptor blocker or an ANG-II type-1receptor blocker (ARBS), include angiocandesartan, eprosartan,irbesartan, losartan and valsartan.

Non-limiting examples of a sympatholytic include a centrally actingsympatholytic or a peripherially acting sympatholytic. Non-limitingexamples of a centrally acting sympatholytic, also known as an centralnervous system (CNS) sympatholytic, include clonidine (catapres),guanabenz (wytensin) guanfacine (tenex) and methyldopa (aldomet).Non-limiting examples of a peripherally acting sympatholytic include aganglion blocking agent, an adrenergic neuron blocking agent, aβ-adrenergic blocking agent or a alpha1-adrenergic blocking agent.Non-limiting examples of a ganglion blocking agent include mecamylamine(inversine) and trimethaphan (arfonad). Non-limiting examples of anadrenergic neuron blocking agent include guanethidine (ismelin) andreserpine (serpasil). Non-limiting examples of a β-adrenergic blockerinclude acenitolol (sectral), atenolol (tenormin), betaxolol (kerlone),carteolol (cartrol), labetalol (normodyne, trandate), metoprolol(lopressor), nadanol (corgard), penbutolol (levatol), pindolol (visken),propranolol (inderal) and timolol (blocadren). Non-limiting examples ofalpha1-adrenergic blocker include prazosin (minipress), doxazocin(cardura) and terazosin (hytrin).

In certain embodiments a cardiovasculator therapeutic agent may comprisea vasodilator (e.g., a cerebral vasodilator, a coronary vasodilator or aperipheral vasodilator). In certain preferred embodiments, a vasodilatorcomprises a coronary vasodilator. Non-limiting examples of a coronaryvasodilator include amotriphene, bendazol, benfurodil hemisuccinate,benziodarone, chloracizine, chromonar, clobenfurol, clonitrate, dilazep,dipyridamole, droprenilamine, efloxate, erythrityl tetranitrane,etafenone, fendiline, floredil, ganglefene, herestrolbis(β-diethylaminoethyl ether), hexobendine, itramin tosylate, khellin,lidoflanine, mannitol hexanitrane, medibazine, nicorglycerin,pentaerythritol tetranitrate, pentrinitrol, perhexyline, pimethylline,trapidil, tricromyl, trimetazidine, trolnitrate phosphate and visnadine.

In certain embodiments, a vasodilator may comprise a chronic therapyvasodilator or a hypertensive emergency vasodilator. Non-limitingexamples of a chronic therapy vasodilator include hydralazine(apresoline) and minoxidil (loniten). Non-limiting examples of ahypertensive emergency vasodilator include nitroprusside (nipride),diazoxide (hyperstat IV), hydralazine (apresoline), minoxidil (loniten)and verapamil.

Non-limiting examples of miscellaneous antihypertensives includeajmaline, γ-aminobutyric acid, bufeniode, cicletainine, ciclosidomine, acryptenamine tannate, fenoldopam, flosequinan, ketanserin, mebutamate,mecamylamine, methyldopa, methyl 4-pyridyl ketone thiosemicarbazone,muzolimine, pargyline, pempidine, pinacidil, piperoxan, primaperone, aprotoveratrine, raubasine, rescimetol, rilmenidene, saralasin, sodiumnitrorusside, ticrynafen, trimethaphan camsylate, tyrosinase andurapidil.

In certain embodiments, an antihypertensive may comprise anarylethanolamine derivative, a benzothiadiazine derivative, aN-carboxyalkyl(peptide/lactam) derivative, a dihydropyridine derivative,a guanidine derivative, a hydrazines/phthalazine, an imidazolederivative, a quanternary ammonium compound, a reserpine derivative or asuflonamide derivative.

Non-limiting examples of arylethanolamine derivatives includeamosulalol, bufuralol, dilevalol, labetalol, pronethalol, sotalol andsulfinalol.

Non-limiting examples of benzothiadiazine derivatives include althizide,bendroflumethiazide, benzthiazide, benzylhydrochlorothiazide,buthiazide, chlorothiazide, chlorthalidone, cyclopenthiazide,cyclothiazide, diazoxide, epithiazide, ethiazide, fenquizone,hydrochlorothizide, hydroflumethizide, methyclothiazide, meticrane,metolazone, paraflutizide, polythizide, tetrachlormethiazide andtrichlormethiazide.

Non-limiting examples of N-carboxyalkyl(peptide/lactam) derivativesinclude alacepril, captopril, cilazapril, delapril, enalapril,enalaprilat, fosinopril, lisinopril, moveltipril, perindopril, quinapriland ramipril.

Non-limiting examples of dihydropyridine derivatives include amlodipine,felodipine, isradipine, nicardipine, nifedipine, nilvadipine,nisoldipine and nitrendipine.

Non-limiting examples of guanidine derivatives include bethanidine,debrisoquin, guanabenz, guanacline, guanadrel, guanazodine,guanethidine, guanfacine, guanochlor, guanoxabenz and guanoxan.

Non-limiting examples of hydrazines/phthalazines include budralazine,cadralazine, dihydralazine, endralazine, hydracarbazine, hydralazine,pheniprazine, pildralazine and todralazine.

Non-limiting examples of imidazole derivatives include clonidine,lofexidine, phentolamine, tiamenidine and tolonidine.

Non-limiting examples of quanternary ammonium compounds includeazamethonium bromide, chlorisondamine chloride, hexamethonium,pentacynium bis(methylsulfate), pentamethonium bromide, pentoliniumtartrate, phenactropinium chloride and trimethidinium methosulfate.

Non-limiting examples of reserpine derivatives include bietaserpine,deserpidine, rescinnamine, reserpine and syrosingopine.

Non-limiting examples of sulfonamide derivatives include ambuside,clopamide, furosemide, indapamide, quinethazone, tripamide and xipamide.

Vasopressors generally are used to increase blood pressure during shock,which may occur during a surgical procedure. Non-limiting examples of avasopressor, also known as an antihypotensive, include amezinium methylsulfate, angiotensin amide, dimetofrine, dopamine, etifelmin, etilefrin,gepefrine, metaraminol, midodrine, norepinephrine, pholedrine andsynephrine.

Non-limiting examples of agents for the treatment of congestive heartfailure include anti-angiotensin II agents, afterload-preload reductiontreatment, diuretics and inotropic agents.

In certain embodiments, an animal subject that can not tolerate anangiotensin antagonist may be treated with a combination therapy. Suchtherapy may combine administration of hydralazine (apresoline) andisosorbide dinitrate (isordil, sorbitrate).

Non-limiting examples of a diuretic include a thiazide orbenzothiadiazine derivative (e.g., althiazide, bendroflumethazide,benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide,chlorothiazide, chlorthalidone, cyclopenthiazide, epithiazide,ethiazide, ethiazide, fenquizone, hydrochlorothiazide,hydroflumethiazide, methyclothiazide, meticrane, metolazone,paraflutizide, polythizide, tetrachloromethiazide, trichlormethiazide),an organomercurial (e.g., chlormerodrin, meralluride, mercamphamide,mercaptomerin sodium, mercumallylic acid, mercumatilin dodium, mercurouschloride, mersalyl), a pteridine (e.g., furtherene, triamterene),purines (e.g., acefylline, 7-morpholinomethyltheophylline, pamobrom,protheobromine, theobromine), steroids including aldosterone antagonists(e.g., canrenone, oleandrin, spironolactone), a sulfonamide derivative(e.g., acetazolamide, ambuside, azosemide, bumetanide, butazolamide,chloraminophenamide, clofenamide, clopamide, clorexolone,diphenylmethane-4,4′-disulfonamide, disulfamide, ethoxzolamide,furosemide, indapamide, mefruside, methazolamide, piretanide,quinethazone, torasemide, tripamide, xipamide), a uracil (e.g.,aminometradine, amisometradine), a potassium sparing antagonist (e.g.,amiloride, triamterene) or a miscellaneous diuretic such as aminozine,arbutin, chlorazanil, ethacrynic acid, etozolin, hydracarbazine,isosorbide, mannitol, metochalcone, muzolimine, perhexyline, ticrnafenand urea.

Non-limiting examples of a positive inotropic agent, also known as acardiotonic, include acefylline, an acetyldigitoxin, 2-amino-4-picoline,amrinone, benfurodil hemisuccinate, bucladesine, cerberosine,camphotamide, convallatoxin, cymarin, denopamine, deslanoside,digitalin, digitalis, digitoxin, digoxin, dobutamine, dopamine,dopexamine, enoximone, erythrophleine, fenalcomine, gitalin, gitoxin,glycocyamine, heptaminol, hydrastinine, ibopamine, a lanatoside,metamivam, milrinone, nerifolin, oleandrin, ouabain, oxyfedrine,prenalterol, proscillaridine, resibufogenin, scillaren, scillarenin,strphanthin, sulmazole, theobromine and xamoterol.

In particular embodiments, an intropic agent is a cardiac glycoside, abeta-adrenergic agonist or a phosphodiesterase inhibitor. Non-limitingexamples of a cardiac glycoside includes digoxin (lanoxin) and digitoxin(crystodigin). Non-limiting examples of a β-adrenergic agonist includealbuterol, bambuterol, bitolterol, carbuterol, clenbuterol,clorprenaline, denopamine, dioxethedrine, dobutamine (dobutrex),dopamine (intropin), dopexamine, ephedrine, etafedrine,ethylnorepinephrine, fenoterol, formoterol, hexoprenaline, ibopamine,isoetharine, isoproterenol, mabuterol, metaproterenol, methoxyphenamine,oxyfedrine, pirbuterol, procaterol, protokylol, reproterol, rimiterol,ritodrine, soterenol, terbutaline, tretoquinol, tulobuterol andxamoterol. Non-limiting examples of a phosphodiesterase inhibitorinclude amrinone (inocor).

Antianginal agents may comprise organonitrates, calcium channelblockers, beta blockers and combinations thereof.

Non-limiting examples of organonitrates, also known asnitrovasodilators, include nitroglycerin (nitro-bid, nitrostat),isosorbide dinitrate (isordil, sorbitrate) and amyl nitrate (aspirol,vaporole).

Endothelin (ET) is a 21-amino acid peptide that has potent physiologicand pathophysiologic effects that appear to be involved in thedevelopment of heart failure. The effects of ET are mediated throughinteraction with two classes of cell surface receptors. The type Areceptor (ET-A) is associated with vasoconstriction and cell growthwhile the type B receptor (ET-B) is associated with endothelial-cellmediated vasodilation and with the release of other neurohormones, suchas aldosterone. Pharmacologic agents that can inhibit either theproduction of ET or its ability to stimulate relevant cells are known inthe art. Inhibiting the production of ET involves the use of agents thatblock an enzyme termed endothelin-converting enzyme that is involved inthe processing of the active peptide from its precursor. Inhibiting theability of ET to stimulate cells involves the use of agents that blockthe interaction of ET with its receptors. Non-limiting examples ofendothelin receptor antagonists (ERA) include Bosentan, Enrasentan,Ambrisentan, Darusentan, Tezosentan, Atrasentan, Avosentan, Clazosentan,Edonentan, sitaxsentan, TBC 3711, BQ 123, and BQ 788.

In certain embodiments, the secondary therapeutic agent may comprise asurgery of some type, which includes, for example, preventative,diagnostic or staging, curative and palliative surgery. Surgery, and inparticular a curative surgery, may be used in conjunction with othertherapies, such as the present invention and one or more other agents.

Such surgical therapeutic agents for vascular and cardiovasculardiseases and disorders are well known to those of skill in the art, andmay comprise, but are not limited to, performing surgery on an organism,providing a cardiovascular mechanical prostheses, angioplasty, coronaryartery reperfusion, catheter ablation, providing an implantablecardioverter defibrillator to the subject, mechanical circulatorysupport or a combination thereof. Non-limiting examples of a mechanicalcirculatory support that may be used in the present invention comprisean intra-aortic balloon counterpulsation, left ventricular assist deviceor combination thereof.

Drug Formulations and Routes for Administration to Subjects

The present invention also provides a pharmaceutical compositioncomprising an agonist or antagonist of miR-29a-c. The agonist may be anexpression vector comprising a nucleic acid segment encoding miR-29a-c,or a polynucleotide comprising a mature miR-29a-c sequence or aneffective portion thereof. The agonist may be comprised in a lipiddelivery vehicle. The antagonist may be a polynucleotide that hybridizesto miR-29a-c or a target thereof.

Where clinical applications are contemplated, pharmaceuticalcompositions will be prepared in a form appropriate for the intendedapplication. Generally, this will entail preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals.

Colloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes, may beused as delivery vehicles for the oligonucleotide inhibitors (e.g.antagonists) of microRNA function or constructs expressing particularmicroRNAs. Commercially available fat emulsions that are suitable fordelivering the nucleic acids of the invention to cardiac and skeletalmuscle tissues include Intralipid®, Liposyn®, Liposyn® II, Liposyn® III,Nutrilipid, and other similar lipid emulsions. A preferred colloidalsystem for use as a delivery vehicle in vivo is a liposome (i.e., anartificial membrane vesicle). The preparation and use of such systems iswell known in the art. Exemplary formulations are also disclosed in U.S.Pat. No. 5,981,505; U.S. Pat. No. 6,217,900; U.S. Pat. No. 6,383,512;U.S. Pat. No. 5,783,565; U.S. Pat. No. 7,202,227; U.S. Pat. No.6,379,965; U.S. Pat. No. 6,127,170; U.S. Pat. No. 5,837,533; U.S. Pat.No. 6,747,014; and WO03/093449, which are herein incorporated byreference in their entireties.

One will generally desire to employ appropriate salts and buffers torender delivery vehicles stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa subject. Aqueous compositions of the present invention comprise aneffective amount of the delivery vehicle comprising the inhibitorpolynucleotides or miRNA polynucleotide sequences (e.g. liposomes orother complexes or expression vectors) or cells, dissolved or dispersedin a pharmaceutically acceptable carrier or aqueous medium. The phrases“pharmaceutically acceptable” or “pharmacologically acceptable” refersto molecular entities and compositions that do not produce adverse,allergic, or other untoward reactions when administered to an animal ora human. As used herein, “pharmaceutically acceptable carrier” includessolvents, buffers, solutions, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike acceptable for use in formulating pharmaceuticals, such aspharmaceuticals suitable for administration to humans. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredients of the present invention, itsuse in therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions, providedthey do not inactivate the vectors or cells of the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention may be via any common route so longas the target tissue is available via that route. This includes oral,nasal, or buccal. Alternatively, administration may be by intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection,or by direct injection into cardiac tissue. Pharmaceutical compositionscomprising miRNA antagonists or expression constructs comprising miRNAsequences may also be administered by catheter systems or systems thatisolate coronary circulation for delivering therapeutic agents to theheart. Various catheter systems for delivering therapeutic agents to theheart and coronary vasculature are known in the art. Some non-limitingexamples of catheter-based delivery methods or coronary isolationmethods suitable for use in the present invention are disclosed in U.S.Pat. No. 6,416,510; U.S. Pat. No. 6,716,196; U.S. Pat. No. 6,953,466, WO2005/082440, WO 2006/089340, U.S. Patent Publication No. 2007/0203445,U.S. Patent Publication No. 2006/0148742, and U.S. Patent PublicationNo. 2007/0060907, which are all herein incorporated by reference intheir entireties. Such compositions would normally be administered aspharmaceutically acceptable compositions, as described supra.

The active compounds may also be administered parenterally orintraperitoneally. By way of illustration, solutions of the activecompounds as free base or pharmacologically acceptable salts can beprepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations generallycontain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use or catheterdelivery include, for example, sterile aqueous solutions or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersions. Generally, these preparations aresterile and fluid to the extent that easy injectability exists.Preparations should be stable under the conditions of manufacture andstorage and should be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. Appropriate solvents ordispersion media may contain, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), suitable mixtures thereof, and vegetable oils. The properfluidity can be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialan antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the activecompounds in an appropriate amount into a solvent along with any otheringredients (for example as enumerated above) as desired, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the desired otheringredients, e.g., as enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation include vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient(s) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions of the present invention generally may be formulated ina neutral or salt form. Pharmaceutically-acceptable salts include, forexample, acid addition salts (formed with the free amino groups of theprotein) derived from inorganic acids (e.g., hydrochloric or phosphoricacids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic,and the like. Salts formed with the free carboxyl groups of the proteincan also be derived from inorganic bases (e.g., sodium, potassium,ammonium, calcium, or ferric hydroxides) or from organic bases (e.g.,isopropylamine, trimethylamine, histidine, procaine and the like.

Upon formulation, solutions are preferably administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations may easily be administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution generally is suitably buffered andthe liquid diluent first rendered isotonic for example with sufficientsaline or glucose. Such aqueous solutions may be used, for example, forintravenous, intramuscular, subcutaneous and intraperitonealadministration. Preferably, sterile aqueous media are employed as isknown to those of skill in the art, particularly in light of the presentdisclosure. By way of illustration, a single dose may be dissolved in 1ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

Cosmetic formulations for increasing collagen deposition in tissues maycomprise at least one antagonist of miR-29a-c. The antagonist may be anantagonist of miR-29a, miR-29b, miR-29c, or combinations thereof. Insome embodiments, the antagonist of miR-29a-c is an antagomir. Theantagonist may be linked or conjugated to agents that facilitate theentry of the antagonist into cell or tissues. Such agents may includecell internalization transporters, such as antennapedia, TAT, BuforinII, Transportan, model amphipathic peptide, K-FGF, Ku70, Prion, pVEC,Pep-1, SynB1, SynB3, SynB5, Pep-7, HN-1,Bis-Guanidinium-Spermidine-Cholesterol,Bis-Guanidinium-Tren-Cholesterol, and polyarginine. The agent may belinked to the miR-29a-c antagonist at its amino or carboxy terminus. Inone embodiment, the agent is linked to the antagonist by a sequence thatis cleaved upon entry to the cell. Such sequences typically compriseconsensus sequences for proteases as are known in the art.

The cosmetic compositions can be formulated into all types of vehicles.Non-limiting examples of suitable vehicles include emulsions (e.g.,water-in-oil, water-in-oil-in-water, oil-in-water, oil-in-water-in-oil,oil-in-water-in-silicone emulsions), creams, lotions, solutions (bothaqueous and hydro-alcoholic), anhydrous bases (such as lipsticks andpowders), gels, and ointments or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art(Remington's, 1990). Variations and other appropriate vehicles will beapparent to the skilled artisan and are appropriate for use in thepresent invention. In certain embodiments, the concentrations andcombinations of the ingredients are selected in such a way that thecombinations are chemically compatible and do not form complexes whichprecipitate from the finished product.

It is also contemplated that aromatic skin-active ingredients andadditional ingredients identified throughout this specification can beencapsulated for delivery to a target area such as skin. Non-limitingexamples of encapsulation techniques include the use of liposomes,vesicles, and/or nanoparticles (e.g., biodegradable andnon-biodegradable colloidal particles comprising polymeric materials inwhich the ingredient is trapped, encapsulated, and/or absorbed—examplesinclude nanospheres and nanocapsules) that can be used as deliveryvehicles to deliver such ingredients to skin (see, e.g., U.S. Pat. No.6,387,398; U.S. Pat. No. 6,203,802; U.S. Pat. No. 5,411,744; and Kreuter1998, which are herein incorporated by reference in their entireties).

Also contemplated are pharmaceutically-acceptable orpharmacologically-acceptable compositions. The phrase“pharmaceutically-acceptable” or “pharmacologically-acceptable” includescompositions that do not produce an allergic or similar untowardreaction when administered to a human. Typically, such compositions areprepared either as topical compositions, liquid solutions orsuspensions, solid forms suitable for solution in, or suspension in,liquid prior to use can also be prepared. Routes of administration canvary with the location and nature of the condition to be treated, andinclude, e.g., topical, inhalation, intradermal, transdermal,parenteral, intravenous, intramuscular, intranasal, subcutaneous,percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion,lavage, direct injection, and oral administration and formulation.

The compositions of the present invention can be incorporated intoproducts. Non-limiting examples of products include cosmetic products,food-based products, pharmaceutical products, etc. By way of exampleonly, non-limiting cosmetic products include sunscreen products, sunlessskin tanning products, hair products, fingernail products, moisturizingcreams, skin benefit creams and lotions, softeners, day lotions, gels,ointments, foundations, night creams, lipsticks, mascaras, eyeshadows,eyeliners, cheek colors, cleansers, toners, masks, or other knowncosmetic products or applications. Additionally, the cosmetic productscan be formulated as leave-on or rinse-off products.

Compositions of the present invention can include additionalingredients. Non-limiting examples of additional ingredients includecosmetic ingredients (both active and non-active) and pharmaceuticalingredients (both active and non-active). The CTFA InternationalCosmetic Ingredient Dictionary and Handbook (2004) describes a widevariety of non-limiting cosmetic ingredients that can be used in thecontext of the present invention. Examples of these ingredient classesinclude: fragrances (artificial and natural), dyes and color ingredients(e.g., Blue 1, Blue 1 Lake, Red 40, titanium dioxide, D&C blue no. 4,D&C green no. 5, D&C orange no. 4, D&C red no. 17, D&C red no. 33, D&Cviolet no. 2, D&C yellow no. 10, and D&C yellow no. 11), adsorbents,emulsifiers, stabilizers, lubricants, solvents, moisturizers (including,e.g., emollients, humectants, film formers, occlusive agents, and agentsthat affect the natural moisturization mechanisms of the skin),water-repellants, UV absorbers (physical and chemical absorbers such asparaminobenzoic acid (“PABA”) and corresponding PABA derivatives,titanium dioxide, zinc oxide, etc.), essential oils, vitamins (e.g., A,B, C, D, E, and K), trace metals (e.g., zinc, calcium and selenium),anti-irritants (e.g., steroids and non-steroidal anti-inflammatories),botanical extracts (e.g., aloe vera, chamomile, cucumber extract, ginkgobiloba, ginseng, and rosemary), anti-microbial agents, antioxidants(e.g., BHT and tocopherol), chelating agents (e.g., disodium EDTA andtetrasodium EDTA), preservatives (e.g., methylparaben andpropylparaben), pH adjusters (e.g., sodium hydroxide and citric acid),absorbents (e.g., aluminum starch octenylsuccinate, kaolin, corn starch,oat starch, cyclodextrin, talc, and zeolite), skin bleaching andlightening agents (e.g., hydroquinone and niacinamide lactate),humectants (e.g., glycerin, propylene glycol, butylene glycol, pentyleneglycol, sorbitol, urea, and manitol), exfoliants (e.g.,alpha-hydroxyacids, and beta-hydroxyacids such as lactic acid, glycolicacid, and salicylic acid; and salts thereof) waterproofing agents (e.g.,magnesium/aluminum hydroxide stearate), skin conditioning agents (e.g.,aloe extracts, allantoin, bisabolol, ceramides, dimethicone, hyaluronicacid, and dipotassium glycyrrhizate), thickening agents (e.g.,substances which that can increase the viscosity of a composition suchas carboxylic acid polymers, crosslinked polyacrylate polymers,polyacrylamide polymers, polysaccharides, and gums), and siliconecontaining compounds (e.g., silicone oils and polyorganosiloxanes).

Pharmaceutical ingredients are also contemplated as being useful withthe emulsion compositions of the present invention. Non-limitingexamples of pharmaceutical ingredients include anti-acne agents, agentsused to treat rosacea, analgesics, anesthetics, anorectals,antihistamines, anti-inflammatory agents including non-steroidalanti-inflammatory drugs, antibiotics, antifungals, antivirals,antimicrobials, anti-cancer actives, scabicides, pediculicides,antineoplastics, antiperspirants, antipruritics, antipsoriatic agents,antiseborrheic agents, biologically active proteins and peptides, burntreatment agents, cauterizing agents, depigmenting agents, depilatories,diaper rash treatment agents, enzymes, hair growth stimulants, hairgrowth retardants including DFMO and its salts and analogs, hemostatics,kerotolytics, canker sore treatment agents, cold sore treatment agents,dental and periodontal treatment agents, photosensitizing actives, skinprotectant/barrier agents, steroids including hormones andcorticosteroids, sunburn treatment agents, sunscreens, transdermalactives, nasal actives, vaginal actives, wart treatment agents, woundtreatment agents, wound healing agents, etc.

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, an individual miRNA is included in a kit. The kitmay further include water and hybridization buffer to facilitatehybridization of the two strands of the miRNAs. In some embodiments, thekit may include one or more oligonucleotides for inhibiting the functionof a target miRNA. The kit may also include one or more transfectionreagent(s) to facilitate delivery of the miRNA or miRNA antagonists tocells.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there is more than one component in the kit (labelingreagent and label may be packaged together), the kit also will generallycontain a second, third or other additional container into which theadditional components may be separately placed. However, variouscombinations of components may be comprised in a vial. The kits of thepresent invention also will typically include a means for containing thenucleic acids, and any other reagent containers in close confinement forcommercial sale. Such containers may include injection or blow-moldedplastic containers into which the desired vials are retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

The container means will generally include at least one vial, test tube,flask, bottle, syringe and/or other container means, into which thenucleic acid formulations are placed, preferably, suitably allocated.The kits may also comprise a second container means for containing asterile, pharmaceutically acceptable buffer and/or other diluent.

The kits of the present invention will also typically include a meansfor containing the vials in close confinement for commercial sale, suchas, e.g., injection and/or blow-molded plastic containers into which thedesired vials are retained.

Such kits may also include components that preserve or maintain themiRNA or miRNA inhibitory oligonucleotides or that protect against theirdegradation. Such components may be RNAse-free or protect againstRNAses. Such kits generally will comprise, in suitable means, distinctcontainers for each individual reagent or solution.

A kit will also include instructions for employing the kit components aswell the use of any other reagent not included in the kit. Instructionsmay include variations that can be implemented. A kit may also includeutensils or devices for administering the miRNA agonist or antagonist byvarious administration routes, such as parenteral or catheteradministration.

It is contemplated that such reagents are embodiments of kits of theinvention. Such kits, however, are not limited to the particular itemsidentified above and may include any reagent used for the manipulationor characterization of miRNA.

Methods for Identifying Modulators

The present invention further comprises methods for identifying agonistsof miR-29a-c that are useful in the prevention or treatment or reversalof cardiac fibrosis, cardiac hypertrophy or heart failure. These assaysmay comprise random screening of large libraries of candidate compounds;alternatively, the assays may be used to focus on particular classes ofcompounds selected with an eye towards structural attributes that arebelieved to make them more likely to promote the expression and/orfunction of miR-29a-c.

To identify a modulator of miR-29a-c, one generally will determine thefunction of a miR-29a-c in the presence and absence of the candidatecompound. For example, a method generally comprises:

-   -   (a) providing a candidate compound;    -   (b) admixing the candidate compound with a miR-29;    -   (c) measuring miR-29a-c activity; and    -   (d) comparing the activity in step (c) with the activity of        miR-29a-c in the absence of the candidate compound,

wherein a difference between the measured activities of miR-29a-cindicates that the candidate compound is, indeed, a modulator ofmiR-29a-c.

Assays also may be conducted in isolated cells, organs, or in livingorganisms.

It will, of course, be understood that all the screening methods of thepresent invention are useful in themselves notwithstanding the fact thateffective candidates may not be found. The invention provides methodsfor screening for such candidates, not solely methods of finding them.

As used herein the term “candidate compound” refers to any molecule thatmay potentially modulate fibrosis- or collagen-regulating aspects ofmiR-29a-c. One will typically acquire, from various commercial sources,molecular libraries that are believed to meet the basic criteria foruseful drugs in an effort to “brute force” the identification of usefulcompounds. Screening of such libraries, includingcombinatorially-generated libraries (e.g., antagomir libraries), is arapid and efficient way to screen a large number of related (andunrelated) compounds for activity. Combinatorial approaches also lendthemselves to rapid evolution of potential drugs by the creation ofsecond, third, and fourth generation compounds modeled on active, butotherwise undesirable compounds.

A quick, inexpensive and easy assay to run is an in vitro assay. Suchassays generally use isolated molecules, can be run quickly and in largenumbers, thereby increasing the amount of information obtainable in ashort period of time. A variety of vessels may be used to run theassays, including test tubes, plates, dishes and other surfaces such asdipsticks or beads.

A technique for high throughput screening of compounds is described inWO 84/03564, which is herein incorporated by reference in its entirety.Large numbers of small antagomir compounds may be synthesized on a solidsubstrate, such as plastic pins or some other surface. Such moleculescan be rapidly screening for their ability to inhibit miR-29a-c.

The present invention also contemplates the screening of compounds fortheir ability to modulate miR-29a-c activity and expression in cells.Various cell lines, including those derived from skeletal muscle cells,can be utilized for such screening assays, including cells specificallyengineered for this purpose. Primary cardiac cells also may be used, ascan the H9C2 cell line.

In vivo assays involve the use of various animal models of heartdisease, musculoskeletal disease, fibrosis, or collagen-loss includingtransgenic animals, that have been engineered to have specific defects,or carry markers that can be used to measure the ability of a candidatesubstance to reach and affect different cells within the organism. Dueto their size, ease of handling, and information on their physiology andgenetic make-up, mice are a preferred embodiment, especially fortransgenics. However, other animals are suitable as well, includingrats, rabbits, hamsters, guinea pigs, gerbils, woodchucks, cats, dogs,sheep, goats, pigs, cows, horses and monkeys (including chimps, gibbonsand baboons). Assays for inhibitors may be conducted using an animalmodel derived from any of these species.

Treatment of animals with test compounds will involve the administrationof the compound, in an appropriate form, to the animal. Administrationwill be by any route that could be utilized for clinical purposes.Determining the effectiveness of a compound in vivo may involve avariety of different criteria, including but not limited to alterationof hypertrophic signaling pathways and physical symptoms of hypertrophy.Also, measuring toxicity and dose responses can be performed in animalsin a more meaningful fashion than in in vitro or in cyto assays.

Transgenic Animals

A particular embodiment of the present invention provides transgenicanimals that lack one or both functional alleles of miR-29a, miR-29b,and/or miR-29c. Also, transgenic animals that express miR-29a-c underthe control of an inducible, tissue selective or a constitutivepromoter, recombinant cell lines derived from such animals, andtransgenic embryos may be useful in determining the exact role thatmiR-29a-c plays in the control of fibrosis and in the development ofpathologic cardiac hypertrophy and heart failure. Furthermore, thesetransgenic animals may provide an insight into heart development. Theuse of an inducible or repressable miR-29a-c encoding nucleic acidprovides a model for over- or unregulated expression. Also, transgenicanimals that are “knocked out” for miR-29a-c, in one or both alleles,are contemplated. Also, transgenic animals that are “knocked out” formiR-29a-c, in one or both alleles for one or both clusters, arecontemplated.

In a general embodiment, a transgenic animal is produced by theintegration of a given transgene into the genome in a manner thatpermits the expression of the transgene. Methods for producingtransgenic animals are generally described by Wagner and Hoppe (U.S.Pat. No. 4,873,191; incorporated herein by reference), and Brinster etal. (1985; incorporated herein by reference).

Typically, a gene flanked by genomic sequences is transferred bymicroinjection into a fertilized egg. The microinjected eggs areimplanted into a host female, and the progeny are screened for theexpression of the transgene. Transgenic animals may be produced from thefertilized eggs from a number of animals including, but not limited toreptiles, amphibians, birds, mammals, and fish.

DNA clones for microinjection can be prepared by any means known in theart. For example, DNA clones for microinjection can be cleaved withenzymes appropriate for removing the bacterial plasmid sequences, andthe DNA fragments electrophoresed on 1% agarose gels in TBE buffer,using standard techniques. The DNA bands are visualized by staining withethidium bromide, and the band containing the expression sequences isexcised. The excised band is then placed in dialysis bags containing 0.3M sodium acetate, pH 7.0. DNA is electroeluted into the dialysis bags,extracted with a 1:1 phenol:chloroform solution and precipitated by twovolumes of ethanol. The DNA is redissolved in 1 ml of low salt buffer(0.2 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) and purified on anElutip-D™ column. The column is first primed with 3 ml of high saltbuffer (1 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) followed by washingwith 5 ml of low salt buffer. The DNA solutions are passed through thecolumn three times to bind DNA to the column matrix. After one wash with3 ml of low salt buffer, the DNA is eluted with 0.4 ml high salt bufferand precipitated by two volumes of ethanol. DNA concentrations aremeasured by absorption at 260 nm in a UV spectrophotometer. Formicroinjection, DNA concentrations are adjusted to 3 μg/ml in 5 mM Tris,pH 7.4 and 0.1 mM EDTA. Other methods for purification of DNA formicroinjection are described in Palmiter et al. (1982); and in Sambrooket al. (2001).

In an exemplary microinjection procedure, female mice six weeks of ageare induced to superovulate with a 5 IU injection (0.1 cc, ip) ofpregnant mare serum gonadotropin (PMSG; Sigma) followed 48 hours laterby a 5 IU injection (0.1 cc, ip) of human chorionic gonadotropin (hCG;Sigma). Females are placed with males immediately after hCG injection.Twenty-one hours after hCG injection, the mated females are sacrificedby C02 asphyxiation or cervical dislocation and embryos are recoveredfrom excised oviducts and placed in Dulbecco's phosphate buffered salinewith 0.5% bovine serum albumin (BSA; Sigma). Surrounding cumulus cellsare removed with hyaluronidase (1 mg/ml). Pronuclear embryos are thenwashed and placed in Earle's balanced salt solution containing 0.5% BSA(EBSS) in a 37.5° C. incubator with a humidified atmosphere at 5% CO₂,95% air until the time of injection. Embryos can be implanted at thetwo-cell stage.

Randomly cycling adult female mice are paired with vasectomized males.C57BL/6 or Swiss mice or other comparable strains can be used for thispurpose. Recipient females are mated at the same time as donor females.At the time of embryo transfer, the recipient females are anesthetizedwith an intraperitoneal injection of 0.015 ml of 2.5% avertin per gramof body weight. The oviducts are exposed by a single midline dorsalincision. An incision is then made through the body wall directly overthe oviduct. The ovarian bursa is then torn with watchmakers forceps.Embryos to be transferred are placed in DPBS (Dulbecco's phosphatebuffered saline) and in the tip of a transfer pipet (about 10 to 12embryos). The pipet tip is inserted into the infundibulum and theembryos transferred. After the transfer, the incision is closed by twosutures.

DEFINITIONS

As used herein, the term “heart failure” is broadly used to mean anycondition that reduces the ability of the heart to pump blood. As aresult, congestion and edema develop in the tissues. Most frequently,heart failure is caused by decreased contractility of the myocardium,resulting from reduced coronary blood flow; however, many other factorsmay result in heart failure, including damage to the heart valves,vitamin deficiency, and primary cardiac muscle disease. Though theprecise physiological mechanisms of heart failure are not entirelyunderstood, heart failure is generally believed to involve disorders inseveral cardiac autonomic properties, including sympathetic,parasympathetic, and baroreceptor responses. The phrase “manifestationsof heart failure” is used broadly to encompass all of the sequelaeassociated with heart failure, such as shortness of breath, pittingedema, an enlarged tender liver, engorged neck veins, pulmonary ralesand the like including laboratory findings associated with heartfailure.

The term “treatment” or grammatical equivalents encompasses theimprovement and/or reversal of the symptoms of heart failure (i.e., theability of the heart to pump blood). “Improvement in the physiologicfunction” of the heart may be assessed using any of the measurementsdescribed herein (e.g., measurement of ejection fraction, fractionalshortening, left ventricular internal dimension, heart rate, etc.), aswell as any effect upon the animal's survival. In use of animal models,the response of treated transgenic animals and untreated transgenicanimals is compared using any of the assays described herein (inaddition, treated and untreated non-transgenic animals may be includedas controls). A compound which causes an improvement in any parameterassociated with heart failure used in the screening methods of theinstant invention may thereby be identified as a therapeutic compound.

The term “dilated cardiomyopathy” refers to a type of heart failurecharacterized by the presence of a symmetrically dilated left ventriclewith poor systolic contractile function and, in addition, frequentlyinvolves the right ventricle.

The term “compound” refers to any chemical entity, pharmaceutical, drug,and the like that can be used to treat or prevent a disease, illness,sickness, or disorder of bodily function. Compounds comprise both knownand potential therapeutic compounds. A compound can be determined to betherapeutic by screening using the screening methods of the presentinvention. A “known therapeutic compound” refers to a therapeuticcompound that has been shown (e.g., through animal trials or priorexperience with administration to humans) to be effective in suchtreatment. In other words, a known therapeutic compound is not limitedto a compound efficacious in the treatment of heart failure.

As used herein, the term “cardiac hypertrophy” refers to the process inwhich adult cardiac myocytes respond to stress through hypertrophicgrowth. Such growth is characterized by cell size increases without celldivision, assembling of additional sarcomeres within the cell tomaximize force generation, and an activation of a fetal cardiac geneprogram. Cardiac hypertrophy is often associated with increased risk ofmorbidity and mortality, and thus studies aimed at understanding themolecular mechanisms of cardiac hypertrophy could have a significantimpact on human health.

As used herein, the term “modulate” refers to a change or an alterationin a biological activity. Modulation may be an increase or a decrease inprotein activity, a change in kinase activity, a change in bindingcharacteristics, or any other change in the biological, functional, orimmunological properties associated with the activity of a protein orother structure of interest. The term “modulator” refers to any moleculeor compound which is capable of changing or altering biological activityas described above.

The term “β-adrenergic receptor antagonist” refers to a chemicalcompound or entity that is capable of blocking, either partially orcompletely, the beta (β) type of adrenoreceptors (i.e., receptors of theadrenergic system that respond to catecholamines, especiallynorepinephrine). Some β-adrenergic receptor antagonists exhibit a degreeof specificity for one receptor subtype (generally β₁); such antagonistsare termed “β₁-specific adrenergic receptor antagonists” and“β₂-specific adrenergic receptor antagonists.” The term β-adrenergicreceptor antagonist” refers to chemical compounds that are selective andnon-selective antagonists. Examples of β-adrenergic receptor antagonistsinclude, but are not limited to, acebutolol, atenolol, butoxamine,carteolol, esmolol, labetolol, metoprolol, nadolol, penbutolol,propanolol, and timolol. The use of derivatives of known β-adrenergicreceptor antagonists is encompassed by the methods of the presentinvention. Indeed any compound, which functionally behaves as aβ-adrenergic receptor antagonist is encompassed by the methods of thepresent invention.

The terms “angiotensin-converting enzyme inhibitor” or “ACE inhibitor”refer to a chemical compound or entity that is capable of inhibiting,either partially or completely, the enzyme involved in the conversion ofthe relatively inactive angiotensin I to the active angiotensin II inthe renin-angiotensin system. In addition, the ACE inhibitorsconcomitantly inhibit the degradation of bradykinin, which likelysignificantly enhances the antihypertensive effect of the ACEinhibitors. Examples of ACE inhibitors include, but are not limited to,benazepril, captopril, enalopril, fosinopril, lisinopril, quiapril andramipril. The use of derivatives of known ACE inhibitors is encompassedby the methods of the present invention. Indeed any compound, whichfunctionally behaves as an ACE inhibitor, is encompassed by the methodsof the present invention.

As used herein, the term “genotypes” refers to the actual geneticmake-up of an organism, while “phenotype” refers to physical traitsdisplayed by an individual. In addition, the “phenotype” is the resultof selective expression of the genome (i.e., it is an expression of thecell history and its response to the extracellular environment). Indeed,the human genome contains an estimated 30,000-35,000 genes. In each celltype, only a small (i.e., 10-15%) fraction of these genes are expressed.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

It is contemplated that any embodiment discussed herein can beimplemented with respect to any method or composition of the invention,and vice versa. Furthermore, compositions and kits of the invention canbe used to achieve methods of the invention.

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Although section headers have been inserted into this application tofacilitate review, such headers should not be construed as a division ofembodiments.

The following examples are included to further illustrate variousaspects of the invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples which followrepresent techniques and/or compositions discovered by the inventor tofunction well in the practice of the invention, and thus can beconsidered to constitute preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentswhich are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

EXAMPLES

Encoded within an intron of the α-MHC gene is miR-208 (FIG. 1A). Likeα-MHC, miR-208 is expressed specifically in the heart with traceexpression in the lung (FIG. 1B). miR-208 is processed out of the α-MHCpre-mRNA rather than being transcribed as a separate transcript.Intriguingly, however, miR-208 displays a remarkably long half-life ofat least 14 days, and can thereby exert functions even when α-MHC mRNAexpression has been down-regulated. Although genetic deletion of miR-208in mice failed to induce an overt phenotype, microarray analysis onhearts from wild-type and miR-208^(−/−) animals at 2 months of agerevealed removal of miR-208 to result in pronounced expression ofnumerous fast skeletal muscle contractile protein genes, which arenormally not expressed in the heart. Thus, these results suggest thatunder normal conditions miR-208 is co-expressed with the solecardiac-specific MHC gene to maintain cardiomyocyte identity byrepressing the expression of skeletal muscle genes in the heart.

The most remarkable function of miR-208 was revealed by the aberrantresponse of miR-208-null mice to cardiac stress (van Rooij et al.,2007). In response to pressure overload by thoracic aortic constrictionor signaling by calcineurin, a calcium/calmodulin-dependent phosphatasethat drives pathological remodeling of the heart, miR-208-null miceshowed virtually no hypertrophy of cardiomyocytes or fibrosis and wereunable to up-regulate β-MHC expression (FIGS. 6-8). In contrast, otherstress responsive genes, such as those encoding ANF and BNP, werestrongly induced in miR-208 mutant animals, demonstrating that miR-208is dedicated specifically to the control of β-MHC expression, which canbe uncoupled from other facets of the cardiac stress response.

β-MHC expression is repressed by thyroid hormone signaling and isup-regulated in the hypothyroid state (Leung et al., 2006).miR-208^(−/−) animals were also resistant to up-regulation of β-MHCexpression following treatment with the T3 inhibitor propylthiouracil(PTU), which induces hypothyroidism. Intriguingly, however, expressionof β-MHC before birth was normal in miR-208 mutant mice, indicating thatmiR-208 is dedicated specifically to the post-natal regulation of β-MHCexpression, which coincides with the acquisition of thyroid hormoneresponsiveness of the β-MHC gene (FIG. 5).

A clue to the mechanism of action of miR-208 comes from the resemblanceof miR-208^(−/−) hearts to hyperthyroid hearts, both of which display ablock to β-MHC expression, up-regulation of stress-response genes andprotection against pathological hypertrophy and fibrosis (FIGS. 6-10).The up-regulation of fast skeletal muscle genes in miR-208^(−/−) heartsalso mimics the induction of fast skeletal muscle fibers in thehyperthyroid state (Wei et al., 2005).

These findings suggest that miR-208 acts, at least in part, byrepressing expression of a common component of stress-response andthyroid hormone signaling pathways in the heart. Among the strongestpredicted targets of miR-208 is the thyroid hormone receptor (TR)co-regulator THRAP1, which can exert positive and negative effects ontranscription (Pantos et al., 2006; Yao and Eghbali, 1992; FIG. 12). TheTR acts through a negative thyroid hormone response element (TRE) torepress β-MHC expression in the adult heart (Zhao et al., 2005). Thus,the increase in THRAP1 expression in the absence of miR-208 would bepredicted to enhance the repressive activity of the TR toward β-MHCexpression, consistent with the blockade to β-MHC expression inmiR-208^(−/−) hearts. However, although THRAP1 appears to be a bone fidetarget for miR-208, these data do not exclude the potential involvementof additional targets in the regulation of β-MHC expression.

Since even a subtle shift towards β-MHC reduces mechanical performanceand efficiency of the adult heart, it would be of therapeutic value toexploit miR-208 regulation to prevent an increase in β-MHC expressionduring cardiac disease. The cardiac specificity and dedication ofmiR-208 to the cardiac stress response, but not to normal cardiacdevelopment, make miR-208 (and its down-stream effectors) an attractivetherapeutic target for manipulating β-MHC levels (FIG. 13).

Materials and Methods

Northern Blot Analysis.

Cardiac tissue samples of left ventricles of anonymous humans diagnosedas having non-failing or failing hearts were obtained from GileadColorado (Westminster, Colo.). Cardiac tissue samples of border zoneregions of anonymous humans diagnosed as having suffered a myocardialinfarction were obtained. Total RNA was isolated from cells, mouse, ratand human cardiac tissue samples or isolated myocytes by using Trizolreagent (Gibco/BRL). Equal loading was confirmed by staining Northerngels with ethidium bromide. Northern blots to detect microRNAs wereperformed as described previously (van rooij et al., 2006). A U6 probeserved as a loading control. To detect α-MHC expression, a Northern blotcontaining 10 μg of RNA from cardiac tissue of both adult wild-type andmiR-208 mutant animals was probed with a cDNA fragment of α-MHC coveringa part of the 5′UTR region and first exon.

PTU Treatment.

Thyroid hormone deficiency was induced by feeding animals for theindicated durations with iodine-free chow supplemented with 0.15% PTUpurchased from Harlan Teklad Co. (TD 97061) (Madison, Wis.).

Microarray and Real-Time PCR Analysis.

Total RNA from cardiac tissue was isolated using Trizol (Invitrogen).Microarray analysis was performed using Mouse Genome 430 2.0 array(Affymetrix). To detect the level of miRNA, RT-PCR was performed usingthe Taqman MicroRNA reverse Transcriptase kit (Applied Biosystems, ABI)according to the manufacturer's recommendations. Five ng of RNA was usedto generate cDNA with a miRNA specific primer, after which a miRNAspecific Taqman probe served to detect the expression level of the miRNAof interest. Following RT-PCR with random hexamer primers (Invitrogen)on RNA samples, the expression of a subset of genes was analyzed byeither PCR or quantitative real time PCR using Taqman probes purchasedfrom ABI.

Generation of miR-208 Mutant Mice.

To generate the miR-208 targeting vector, a 0.4 kb fragment (5′ arm)extending upstream of the miR-208 coding region was digested with SacIIand NotI and ligated into the pGKneoF2L2dta targeting plasmid upstreamof the loxP sites and the Frt-flanked neomycin cassette. A 3.3 kbfragment (3′ arm) was digested with SalI and HindIII and ligated intothe vector between the neomycin resistance and Dta negative selectioncassettes. Targeted ES-cells carrying the disrupted allele wereidentified by Southern blot analysis with 5′ and 3′ probes. ThreemiR-208 targeted ES clones were identified and used for blastocystinjection. The resulting chimeric mice were bred to C57BL/6 to obtaingermline transmission of the mutant allele.

Generation of Transgenic Mice.

A mouse genomic fragment flanking the miRNA of interest was subclonedinto a cardiac-specific expression plasmid containing the α-MHC andhuman GH poly(A)+ signal (Kiriazis and Kranias, 2000). Genomic DNA wasisolated from mouse tail biopsies and analyzed by PCR using primersspecific for the human GH poly(A)+ signal.

Western Blotting.

Myosin was extracted from cardiac tissue as described (Morkin, 2000).MHC isoforms were separated by SDS PAGE and Western blotting wasperformed with mouse monoclonal α-MHC (BA-G5) (ATCC, Rockville, Md.) andmouse monoclonal antimyosin (slow, skeletal M8421) (Sigma, Mo.), whichis highly specific for β-MHC. To detect all striated myosin a panspecific antibody (mouse monoclonal 3-48; Accurate Chemical & ScientificCorporation, NY) was used. THRAP1 was detected by immunoprecipitationfrom 400 μg of cardiac protein lysate. After pre-clearing the samplesfor 1 hour at 4° C., the supernatant was incubated overnight at 4° C.with 1 μl rabbit polyclonal anti-THRAP1 (a kind gift of R. Roeder,Rockefeller University) and 15 μl of protein A beads. The beads werewashed three times with lysis buffer and boiled in SDS sample buffer.Immunoprecipitated THRAP1 protein was resolved by SDS-PAGE and analyzedusing rabbit polyclonal anti-THRAP1 at a dilution of 1:3000 andanti-rabbit IgG conjugated to horseradish peroxidase at a dilution of1:5000 with detection by Luminol Reagent (Santa Cruz).

Histological Analysis and RNA In Situ Hybridization.

Tissues used for histology were incubated in Krebs-Henselheit solution,fixed in 4% paraformaldehyde, sectioned, and processed for hematoxylinand eosin (H&E) and Masson's Trichrome staining or in situ hybridizationby standard techniques (Krenz and Robbins, 2004). ³⁵5-labeled RNA probeswere generated using Maxiscript kit (Amersham). Signals werepseudocolored in red using Adobe Photoshop.

Transthoracic Echocardiography.

Cardiac function and heart dimensions were evaluated by two-dimensionalechocardiography in conscious mice using a Vingmed System (GE VingmedUltrasound, Horten, Norway) and a 11.5-MHz linear array transducer.M-mode tracings were used to measure anterior and posterior wallthicknesses at end diastole and end systole. Left ventricular (LV)internal diameter (LVID) was measured as the largest anteroposteriordiameter in either diastole (LVIDd) or systole (LVIDs). The data wereanalyzed by a single observer blinded to mouse genotype. LV fractionalshortening (FS) was calculated according to the following formula: FS(%)=[(LVIDd-LVIDs)/LVIDd]×100.

Plasmids and Transfection Assays.

A 305 bp genomic fragment encompassing the miR-208 coding region wasamplified by PCR and ligated into pCMV6. A 1 kb fragment encompassingthe entire murine THRAP1-UTR was PCR-amplified and ligated into anHA-tagged pCMV6 expression construct and the firefly luciferase (f-luc)reporter construct (pMIR-REPORT™, Ambion). A mutation of the UCGUCUUAmiR-208 seed binding sequence was constructed through PCR-basedmutagenesis.

Cell Culture, Transfection and Luciferase Assays.

A 1793-bp genomic fragment encompassing miR-29b-1 and miR-29a codingregion was amplified by PCR and ligated into pCMV6. Genomic fragments ofthe murine 3′UTR encompassing the miR-29a-c binding site(s) werePCR-amplified and ligated into the firefly luciferase (f-luc) reporterconstruct (pMIR-REPORT™, Ambion). COS cells were transfected with Fugene6 (Stratagene) according to manufacturer's instructions. The totalamount of DNA per well was kept constant by adding the correspondingamount of expression vector without a cDNA insert. 48 hours aftertransfection, cell extracts were assayed for luciferase expression usingthe luciferase assay kit (Promega). Relative promoter activities areexpressed as luminescence relative units normalized for β-galactosidaseexpression in the cell extracts.

Cardiac fibroblasts (CFs) were isolated as described previously (Simpsonand Savion, 1982). Briefly, hearts were excised from anesthetizedneonatal 1-2 day-old Sprague-Dawley rats (Harlan Sprague Dawley,Indianapolis, Ind.), minced, and digested with pancreatin 0.1%. Cellswere plated on primaria plates for 2 h, and the medium which containedthe cardiomyocyte fraction of the digested tissue was removed. Cardiacfibroblasts attached and proliferated much more rapidly than cardiacmyocytes; this produced virtually pure fibroblast cultures after thefirst passage, which was confirmed by repeated differential plating andmicroscopic evaluation. Cells were detached with 0.05% trypsin forpassaging, and culture studies were performed at passages 2 to 4. Cellswere grown in high glucose (4.5 gm/lt) Dulbecco's modified Eagle'smedium (DMEM) containing 10% heat-inactivated FBS and antibiotics(Penicillin and streptomycin). Myofibroblast differentiation was inducedby changing the medium to low serum (2% FBS) with L-ascorbic acid (10μg/μl) and administration of 10 ng/ml TGFβ1 for 48 hours.

In Vivo miR-29b Silencing by Anti-miR Treatment.

Chemically modified antisense oligonucleotides comprising a sequencecomplementary to miR-29b (anti-miR-29b) were used to inhibit miR-29bexpression. All bases were 2′-OMe modified, the first two and last fourbases contained a phosphorothioate internucleoside bond and the 3′ endof the oligonucleotides was conjugated to cholesterol. Eight week-oldC57BL/6 male mice received either anti-miR-29b(AsAsCACUGAUUUCAAAUGGUsGsCsUsAs-Cholesterol) or mismatch miR-29b(AsAsAACUGAUGUCACAUGGUsGsAsUsAs-Cholesterol) at a dose of 80 mg/kg bodyweight or a comparable volume of saline through tail vein injection.Tissues were collected either 3 days or 3 weeks after treatment.

Example 1 Regulation of Cardiac Hypertrophy and Heart Failure byStress-Responsive miRNAs

In light of their involvement in modulating cellular phenotypes, theinventors hypothesized that miRNAs may play a role in regulating theresponse of the heart to cardiac stress, which is known to result intranscriptional and translational changes in gene expression. Toinvestigate the potential involvement of miRNAs in cardiac hypertrophy,they performed a side-by-side miRNA microarray analysis in 2 establishedmouse models of cardiac hypertrophy, using a microarray that represented186 different miRNAs (Babak et al., 2004). Mice that were subjected tothoracic aortic banding (TAB), which induces hypertrophy by increasedafterload on the heart (Hill et al., 2000), were compared to shamoperated animals. In a second model, transgenic mice expressingactivated calcineurin (CnA) in the heart, which results in a severe,well-characterized form of hypertrophy (Molkentin et al., 1998), werecompared to wild-type littermates (FIG. 14A). RNA isolated from heartsof mice subjected to TAB showed increased expression of 27 miRNAscompared to sham-operated controls, and CnA Tg mice showed increasedexpression of 33 miRNAs compared with non-transgenic littermatecontrols, of which 21 were up-regulated in both models. Similarly, TABand CnA-induced hypertrophy were accompanied by reduced expression of 15and 14 miRNAs, respectively, of which 7 miRNAs were down-regulated incommon (FIG. 14B). Northern analysis of these miRNAs (unpublished data)and previous microarray analyses (Barad et al., 2004; Sempere et al.,2004; Shingara et al., 2005; Liu et al., 2004) indicate that they areexpressed in a wide range of tissues. Based on their relative expressionlevels, conservation of human, rat and mouse sequences, and levels ofexpression during hypertrophy, the inventors focused on 11 up- and 5down-regulated miRNAs (FIG. 14C).

Northern blot analysis of cardiac RNA from WT and CnA Tg animalsconfirmed an increased expression of miRs-21, -23, -24, -125b, -195,-199a, and -214, and decreased expression of miRs-29, -93, -150 and-181b (FIG. 14C and FIG. 15). Collectively, these data indicate thatdistinct miRNAs are regulated during cardiac hypertrophy, suggesting thepossibility that they function as modulators of this process.

Example 2 Discovery of the miR-29 Family as Down-Stream Targets forRegulation by miR-208

The inventors performed a miRNA microarray on hearts from wild-type andmiR-208-null mice in an effort to identify downstream miRNAs that mightmediate the actions of miR-208 (FIG. 16). They discovered that multiplemembers of the miR-29 family were up-regulated in miR-208-null mice(FIG. 17). Target prediction indicated that miR-29 family memberstargeted mRNAs encoding multiple collagens and other components of theextracellular matrix (FIG. 18). Thus, the up-regulation of miR-29 familymembers in miR-208-null mice is likely to account for the block tofibrosis seen in these animals (FIG. 19).

The discovery that miR-29a-c is down-regulated in the diseased heart andtargets mRNAs encoding collagens and extracellular matrix proteinssuggests that strategies to enhance expression of miR-29a-c or itsassociation with target mRNAs can have beneficial effects on the heartin the settings of pathological cardiac remodeling and fibrosis.Moreover, elevation of miR-29a-c expression or function may preventfibrosis associated with many diseases in tissues such as skeletalmuscle, liver, lung, kidney and others. In addition, the discovery thatmiR-208 represses miR-29a-c expression, and that loss of miR-208upregulates miR-29a-c expression, indicates that miR-29a-c is adownstream mediator of the actions of miR-208 on the heart.

Example 3 MiR-29a-c Regulates the Expression of Fibrotic Genes

To begin to define the possible functions for miR-29a-c in the heartfollowing MI, the inventors made use of computational predictions toidentify possible miR-29a-c targets. The Targetscan prediction websiteindicated an unexpectedly high number of fibrosis-related mRNAs encodingcollagens, metallopeptidases, and integrins as possible targets formiR-29a-c (word-wide web at targetscan.org). To determine whether thedownregulation of miR-29a-c might regulate cardiac fibrosis, theinventors focused on predicted targets implicated in ECM production inheart. Elastin (ELN), fibrillin 1 (FBN1), collagen type I, α1 and α2(COL1A1, COL1A2) and collagen type III, α1 (COL3A1) all contain one ormore conserved potential seed sequences for miR-29a-c (FIG. 20A).

Because miRNAs down-regulate the steady state levels, as well as thetranslation, of their target mRNAs, the inventors analyzed theexpression of predicted miR-29a-c mRNA targets. Real-time RT-PCRanalysis of these key regulatory genes for cardiac fibrosis in cardiacsamples 3 days after MI indicated that the specific downregulation ofmiR-29a-c in the infarcted region correlates with the increase inexpression of COL1A1, COL1A2, COL3A1, and FBN1. In contrast, ELNappeared unchanged in the border zone, and even showed an increase inthe remote myocardium (FIG. 20B).

Using a CMV-driven expression plasmid, the inventors overexpressedmiR-29b-1 and miR-29a in COS cells (FIG. 20C) with luciferase expressionplasmids containing the 3′-UTRs of the predicted miR-29a-c targets.Increasing amounts of CMV-driven miR-29b-1/miR-29a resulted in adose-dependent decrease in luciferase activity, while comparable amountsof miR-206, as a control, had no effect (FIGS. 20C-D), substantiatingthese mRNAs as targets for repression by miR-29a-c.

Example 4 Regulation of miR-29a-c in Cardiac Fibroblasts

Cardiac fibrosis is a major aspect of the remodeling process typicallyseen in the failing heart. The proliferation of fibroblasts andincreased deposition of ECM components results in myocardial stiffnessand diastolic dysfunction. Transforming growth factor β (TGFβ) has beenshown to play a dominant role in the production and deposition ofcollagens in the heart and induces a transformation of fibroblasts intomyofibroblasts (Border and Noble, 1994). Real-time PCR analysis oncardiac fibroblasts exposed to TGFβ revealed a decrease in miR-29a-cexpression, suggesting that the decrease in miR-29a-c following MI mightbe TGFβ-regulated (FIG. 21A). Interestingly, natriuretic peptides likeB-type natriuretic peptide (BNP) have been shown to inhibitTGFβ-regulated gene expression related to fibrosis and myofibroblastconversion (Kapoun et al., 2004). In this regard, the inventors reportedpreviously that mice lacking the cardiac-specific miRNA miR-208 wereresistant to cardiac fibrosis and remodeling and exhibited increasedexpression of BNP at baseline (van Rooij et al., 2007). Since BNP isknown to antagonize the effects of TGFβ, the inventors speculated thatthe increased levels of BNP in these mice might enhance the expressionof miR-29a-c. Indeed, Northern analysis showed a dose-dependent increasein miR-29a-c expression upon removal of miR-208, which coincided with anincreasing expression level of BNP (FIG. 21B). These data indicate thatTGFβ induces the expression of collagen related genes in fibroblasts atleast partly through decreasing the level of miR-29a-c, which can beinhibited by BNP secreted by cardiomyocytes.

Example 5 In Vivo Knockdown of miR-29a-c Induces Fibrosis and Expressionof Collagen Genes

To further explore the potential role of miR-29a-c as a negativeregulator of collagen expression, the inventors knocked down miR-29b invivo using cholesterol-modified oligonucleotides complementary to themature miRNA sequence of miR-29b (anti-miR-29b) and either saline or anoligonucleotide containing a four-base mismatch (mm miR-29b) as anegative control (FIG. 22A). Three days after a single tail veininjection of anti-miR-29b (80 mg/kg), the inventors observed a dramaticdiminution of miR-29b expression in all tissues examined (FIG. 22B). Incontrast, a comparable dose of the mm miR-29b antisense oligonucleotidehad no effect on the expression level of miR-29b compared to the salinecontrol. Knockdown by anti-miR-29b appeared to be specific to the maturemiRNA, since the level of pre-miRNA remained comparable between anti-miRand mm treated animals. While the knockdown in the liver and kidneyappeared to be complete, a low level of miR-29b remained detectable inthe heart and lung (FIG. 22B).

Since the other miR-29 members share high sequence homology withmiR-29b, the expression of miR-29a and -c in response to anti-miR-29bwas also examined. While a significant knockdown in liver and kidney(especially for miR-29c), was detected, cardiac expression did notappear to change (FIG. 23). Real-time PCR analysis indicated thatmiR-29b knockdown was sufficient to induce the expression of collagengenes in the liver specifically, while this effect was absent in themismatch controls (FIG. 22C).

To enhance cardiac knockdown of miR-29b, the inventors injected 80 mg/kgof oligonucleotide intravenously on two consecutive days and collectedmaterial 3 weeks later. Northern analysis indicated complete knockdownof miR-29b in kidney and liver in response to anti-miR-29b compared tothe expression level seen after mm miR-29b injection. Cardiac levels ofmiR-29b were also dramatically reduced, while the expression of miR-29bin lung appeared unaffected by anti-miR-29b (FIG. 22D). Collagenexpression in the heart was increased in response to miR-29b inhibition(FIG. 22E). Taken together, these data indicate that miR-29b functionsas a negative regulator of collagen gene expression in vivo and therebyinfluences collagen deposition and fibrosis in the heart and liver.

Example 6 Down-Regulation of Collagen Expression with a miR-29a-c Mimic

To determine whether overexpression of miR-29a-c was capable of reducingcollagen expression, the inventors exposed fibroblasts to a miR-29bmimic. The level of miR-29b expression in fibroblasts cultures increasedby as much as 400-fold after 3 days of exposure to miR-29b mimic (FIG.22F). miR-29a expression was unaffected and miR-29c expression wasincreased only slightly by miR-29b mimic (FIG. 22F). Real-time PCRanalysis indicated that the expression of collagen genes was diminishedin response to miR-29b mimic (FIG. 22G). However, the magnitude of thedecrease in collagen expression was modest compared to the increase inexpression of miR-29b, indicating that miR-29a-c levels are not the soledeterminant of collagen levels.

All publications, patents and patent applications discussed and citedherein are incorporated herein by reference in their entireties. All ofthe compositions and methods disclosed and claimed herein can be madeand executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods, and in the steps or in the sequence of stepsof the methods described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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The invention claimed is:
 1. A method of inducing collagen deposition ina tissue of a subject in need thereof comprising contacting said tissuewith an antisense oligonucleotide comprising a sequence that is at leastpartially complementary to a miR-29a, miR-29b, and/or miR-29c sequence.2. The method of claim 1, wherein the antisense oligonucleotidecomprises a sequence that is at least partially complementary to SEQ IDNO: 18, SEQ ID NO: 19, and/or SEQ ID NO:
 20. 3. The method of claim 2,wherein the antisense oligonucleotide comprises a sequence that is atleast 85% complementary to SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ IDNO:
 20. 4. The method of claim 2, wherein the antisense oligonucleotidecomprises a sequence that is at least 95% complementary to SEQ ID NO:18, SEQ ID NO: 19, and/or SEQ ID NO:
 20. 5. The method of claim 2,wherein the antisense oligonucleotide comprises a sequence that is 100%complementary to SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO:
 20. 6.The method of claim 1, wherein the antisense oligonucleotide comprises asequence that is substantially complementary to a pre-miR-29a,pre-miR-29b, or pre-miR-29c sequence.
 7. The method of claim 1, whereinthe antisense oligonucleotide is about 15 to about 50 nucleotides inlength.
 8. The method of claim 1, wherein the antisense oligonucleotideis about 19 to about 25 nucleotides in length.
 9. The method of claim 1,wherein the antisense oligonucleotide comprises at least one sugarand/or backbone modification.
 10. The method of claim 9, wherein thesugar modification is a modification selected from the group consistingof 2′-O-alkyl, 2′-O-methyl, 2′-O-methoxyethyl, 2′-fluoro, and a lockednucleic acid.
 11. The method of claim 9, wherein the backbonemodification is a phosphorothioate linkage.
 12. The method of claim 1,wherein the antisense oligonucleotide is conjugated to cholesterol atits 3′ terminus.
 13. The method of claim 1, wherein said tissue isfacial tissue.
 14. The method of claim 13, wherein said facial tissue isa forehead, a lip, a cheek, a chin, an eyebrow, an eyelid, under theeye, or near the mouth.
 15. The method of claim 1, wherein said tissueis skin, bone, or blood vessels.
 16. The method of claim 1, wherein saidtissue comprises a wound, a skin graft, scar tissue, wrinkles, lax skin,sun damage, chemical damage, heat damage, cold damage, and/or stretchmarks.
 17. The method of claim 1, wherein said contacting comprisesinjection of the antisense oligonucleotide into said tissue orvasculature that feeds said tissue.
 18. The method of claim 1, whereinsaid contacting comprises applying a topical formulation comprising theantisense oligonucleotide.
 19. The method of claim 18, wherein saidtopical formulation is an ointment, cream, gel, salve, or balm.
 20. Themethod of claim 1, further comprising administering a second agent orsecond treatment to the subject.
 21. The method of claim 20, whereinsaid second agent is topical vitamin A, topical vitamin C, or vitamin E.22. The method of claim 20, wherein said second treatment comprises achemical peel, laser treatment, dermaplaning, or dermabrasion.